Bioproduction of para-hydroxycinnamic acid

ABSTRACT

The present invention provides several methods for biological production of para-hydroxycinnamic acid (PHCA). The invention is also directed to the discovery of new fungi and bacteria that possess the ability to convert cinnamate to PHCA. The invention relates to developing of a new biocatalyst for conversion of glucose to PHCA by incorporation of the wild type PAL from the yeast  Rhodotorula glutinis  into  E. coli  underlining the ability of the wildtype PAL to convert tyrosine to PHCA. The invention is also directed to developing a new biocatalyst for conversion of glucose to PHCA by incorporation of the wildtype PAL from the yeast  Rhodotorula glutinis  plus the plant cytochrome P-450 and the cytochrome P-450 reductase into  E. coli . In yet another embodiment, the present invention provides for the developing of a new biocatalyst through mutagenesis of the wild type yeast PAL which possesses enhanced tyrosine ammonia-lyase (TAL) activity.

This application claims benefit of Provisional Application No.60/147,719 filed Aug. 6, 1999.

FIELD OF THE INVENTION

This invention relates to the field of molecular biology andmicrobiology. More specifically, this invention describes a new,genetically engineered biocatalyst possessing enhanced tyrosineammonia-lyase activity.

BACKGROUND OF THE INVENTION

Phenylalanine ammonia-lyase (PAL) (EC 4.3.1.5) is widely distributed inplants (Koukol et al., J. Biol. Chem. 236:2692-2698 (1961)), fungi(Bandoni et al., Phytochemistry 7:205-207 (1968)), yeast (Ogata et al.,Agric. Biol. Chem. 31:200-206 (1967)), and Streptomyces (Emes et al.,Can. J. Biochem. 48:613-622 (1970)), but it has not been found inEscherichia coli or mammalian cells (Hanson and Havir In The Enzymes,3^(rd) ed.; Boyer, P., Ed.; Academic: New York, 1967; pp 75-167). PAL isthe first enzyme of phenylpropanoid metabolism and catalyzes the removalof the (pro-3S)-hydrogen and −NH₃ ⁺ from L-phenylalanine to formtrans-cinnamic acid. In the presence of a P450 enzyme system,trans-cinnamic acid can be converted to para-hydroxycinnamic acid (PHCA)which serves as the common intermediate in plants for production ofvarious secondary metabolites such as lignin and isoflavonoids. Inmicrobes however, cinnamic acid and not the PHCA acts as the precursorfor secondary metabolite formation. No cinnamate hydroxylase enzyme hasso far been characterized from microbial sources. The PAL enzyme inplants is thought to be a regulatory enzyme in the biosynthesis oflignin, isoflavonoids and other phenylpropanoids (Hahlbrock et al.,Annu. Rev. Plant Phys. Plant Mol. Biol. 40:347-369 (1989)). However, inthe red yeast, Rhodotorula glutinis (Rhodosporidium toruloides), thislyase degrades phenylalanine as a catabolic function and the cinnamateformed by the action of this enzyme is converted to benzoate and othercellular materials.

The gene sequence of PAL from various sources, including Rhodosporidiumtoruloides, has been determined and published (Edwards et al., Proc.Natl. Acad. Sci., USA 82:6731-6735 (1985); Cramer et al., Plant Mol.Biol. 12:367-383 (1989); Lois et al., EMBO J. 8:1641-1648 (1989); Minamiet al., Eur. J. Biochem. 185:19-25 (1989); Anson et al., Gene 58:189-199(1987); Rasmussen & Oerum, DNA Sequence, 1:207-211 (1991). The PAL genesfrom various sources have been over-expressed as active PAL enzyme inyeast, Escherichia coli and insect cell culture (Faulkner et al., Gene143:13-20 (1994); Langer et al., Biochemistry 36:10867-10871 (1997);McKegney et al., Phytochemistry 41:1259-1263 (1996)). PAL has receivedattention because of its potential usefulness in correcting the inbornerror of metabolism phenylketonuria (Bourget et al., FEBS Lett. 180:5-8(1985); U.S. Pat. No. 5,753,487), in altering tumor metabolism (Fritz etal. J. Biol. Chem. 251:4646-4650 (1976)), in quantitative analysis ofserum phenylalanine (Koyama et al., Clin. Chim. Acta, 136:131-136(1984)) and as a route for synthesizing L-phenylalanine from cinnamicacid (Yamada et al., Appl. Environ. Microbiol. 42:773 (1981), Hamiltonet al., Trends in Biotechnol. 3:64-68 (1985) and Evans et al., MicrobialBiotechnology 25:399-405 (1987)).

In plants, the PAL enzyme converts phenylalanine to trans-cinnamic acidwhich in turn is hydroxylated at the para position bycinnamate-4-hydroxylase to make PHCA (Pierrel et al., Eur. J. Biochem.224:835 (1994); Urban et al., Eur. J Biochem. 222:843 (1994);Cabello-Hurtado et al., J. Biol. Chem. 273:7260 (1998); and Teutsch etal., Proc. Natl. Acad. Sci. USA 90:4102 (1993)). However, since furthermetabolism of cinnamic acid in microbial systems does not usuallyinvolve its para hydroxylation to PHCA, information regarding thisreaction in microorganisms is scarce.

Information available indicates that PAL from some plants andmicro-organisms, in addition to its ability to convert phenylalanine tocinnamate, can accept tyrosine as substrate. In such reactions theenzyme activity is designated tyrosine ammonia lyase (TAL). Conversionof tyrosine by TAL results in the direct formation of PHCA from tyrosinewithout the intermediacy of cinnamate. However, all natural PAL/TALenzymes prefer to use phenylalanine rather than tyrosine as theirsubstrate. The level of TAL activity is always lower than PAL activity,but the magnitude of this difference varies over a wide range. Forexample, the parsley enzyme has a K_(M) for phenylalanine of 15-25 μMand for tyrosine 2.0-8.0 mM with turnover numbers 22/sec and 0.3/secrespectively (Appert et al., Eur. J. Biochem. 225:491 (1994)). Incontrast, the maize enzyme has a K_(M) for phenylalanine only fifteentimes higher than for tyrosine, and turnover numbers about ten-foldhigher (Havir et al., Plant Physiol. 48:130 (1971)). The exception tothis rule, is the yeast, Rhodosporidium, in which a ratio of TALcatalytic activity to PAL catalytic activity is approximately 0.58(Hanson and Havir In The Biochemistry of Plants; Academic: New York,1981; Vol. 7, pp 577-625).

The above mentioned biological systems provide a number of enzymes thatmay be useful in the production of PHCA, however, the efficientproduction of this monomer has not been achieved. The problem to beovercome therefore is the design and implementation of a method for theefficient production of PHCA from a biological source using aninexpensive substrate or fermentable carbon source. Applicants havesolved the stated problem by engineering both microbial and plant hoststo produce PHCA, either by the overexpression of foreign genes encodingPAL and p450/p-450 reductase system or by the expression of genesencoding mutant and wildtype TAL activity.

SUMMARY OF THE INVENTION

The object of the present invention is bioproduction of PHCA, a compoundthat has potential as a monomer for production of Liquid CrystalPolymers (LCP). There are two potential bio-routes for production ofPHCA from glucose and other fermentable carbon substrates:

1) Conversion of phenylalanine to cinnamic acid to PHCA. This routerequires the enzyme PAL as well as a cytochrome P-450 and a cytochromeP-450 reductase (Scheme 1).

2) Conversion of tyrosine to PHCA in one step without the intermediacyof cinnamate (Scheme 1). This route requires the enzyme TAL which islikely to be very similar to PAL but with a higher substrate specificityfor tyrosine. This route does not require the cytochrome P-450 and thecytochrome P450 reductase. Operation of the TAL route therefore requiresgeneration of a biocatalyst with increased TAL activity to functionthrough the TAL route.

The present invention describes methods for bioproduction of PHCAthrough conversion of: 1) cinnamate to PHCA; 2) glucose to phenylalanineto PHCA via the PAL route and 3) through generation of a new biocatalystpossessing enhanced tyrosine ammonia-lyase (TAL) activity. The evolutionof TAL requires isolation of a yeast PAL gene, mutagenesis and evolutionof the PAL coding sequence, and selection of variants with improved TALactivity. The instant invention further demonstrates the bioproductionof PHCA from glucose through the above mentioned routes in various fungiand bacteria.

It is an object of the present invention therefore to provide a methodfor the production of PHCA comprising: (i) contacting a recombinant hostcell with a fermentable carbon substrate, said recombinant cell lackinga P-450/P-450 reductase system and comprising a gene encoding a tyrosineammonia lyase activity operably linked to suitable regulatory sequences(ii) growing said recombinant cell for a time sufficient to producePHCA; and (iii) optionally recovering said PHCA. Within the context ofthe invention a fermentable carbon substrate may be selected from thegroup consisting of monosaccharides, oligosaccharides, polysaccharides,carbon dioxide, methanol, formaldehyde, formate, and carbon-containingamines and the host cell from the group consisting of bacteria, yeasts,filamentous fungi, algae and plant cells.

Similarly provided are recombinant host cells lacking a cytochromeP-450/P-450 reductase system and comprising a gene encoding a tyrosineammonia lyase activity operably linked to suitable regulatory sequences.

Additionally provided is a method for the production of PHCA comprising:(i) contacting a recombinant yeast cell with a fermentable carbonsubstrate, said recombinant cell comprising: a) a gene encoding a plantP-450/P-450 reductase system; and b) a gene encoding a yeast PALactivity operably linked to suitable regulatory sequences; (ii) growingsaid recombinant cell for a time sufficient to produce PHCA; and (iii)optionally recovering said PHCA.

It is another object of the present invention to provide a method foridentifying a gene encoding a TAL activity comprising: (i) contacting arecombinant microorganism comprising a foreign gene suspected ofencoding a TAL activity with PHCA for a time sufficient to metabolizePHCA; and (ii) monitoring the growth the recombinant microorganismwhereby growth of the organism indicates the presence of a gene encodinga TAL activity.

Similarly a method for identifying a gene encoding a TAL activity isprovided comprising: (i) transforming a host cell capable of using PHCAas a sole carbon source with a gene suspected of encoding a TAL activityto create a transformant; (ii) comparing the rate of growth of thetransformant with an untransformed host cell capable of using PHCA as asole carbon source wherein an accelerated rate of growth by thetransformant indicates the presence of a gene encoding a TAL activity.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE DESCRIPTIONS ANDBIOLOGICAL DEPOSITS

FIG. 1 is a plasmid map of the vector PCA12Km, derived from pBR322, andused for the construction of the PAL expression vector PCA18Km.

FIG. 2 is a plasmid map of the vector pETAL containing the mutal PAL/TALenzyme.

FIG. 3 shows the SDS-PAGE of purified mutant PAL enzyme and the cellcrude extracts used as the starting materials for purification of themutant PAL enzyme.

FIG. 4 is a plasmid map of the expression vector pGSW18 used for theexpression of the mutant PAL/TAL enzyme in yeast.

Applicants made the following biological deposits under the terms of theBudapest Treaty on the International Recognition of the Deposit ofMicro-organisms for the Purposes of Patent Procedure at the AmericanType Culture Collection (ATCC) 10801 University Boulevard, Manassas, Va.20110-2209:

International Depositor Identification Depository Reference DesignationDate of Deposit E. coli pKK223-3 PAL in DH10B PTA 407 July 21, 1999 S.cereviseae containing wild-type PAL PTA 408 July 21, 1999 S. cereviseaeAro4GSW PTA 409 July 21, 1999

The invention can be more fully understood from the following detaileddescription and the accompanying sequence descriptions which form a partof this application.

Applicant(s) have provided 14 sequences in conformity with 37 C.F.R.1.821-1.825 (“Requirements for Patent Applications Containing NucleotideSequences and/or Amino Acid Sequence Disclosures—the Sequence Rules”)and consistent with World Intellectual Property Organization (WIPO)Standard ST.25 (1998) and the sequence listing requirements of the EPOand PCT (Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of theAdminstrative Instructions).

SEQ ID NOs:1-6 are primers used for vector construction.

SEQ ID NO:7 is the nucleotide sequence encoding the wildtype R. glutinisPAL enzyme.

SEQ ID NO:8 is the deduced amino acid sequence encoded by the nucleotidesequence encoding the wildtype R. glutinis PAL enzyme.

SEQ ID NO:9 is the nucleotide sequence encoding the mutant R. glutinisPAL enzyme having enhanced TAL activity.

SEQ ID NO:10 is the deduced amino acid sequence encoded by thenucleotide sequence encoding the mutant R. glutinis PAL enzyme havingenhanced TAL activity.

SEQ ID NO:11 is the nucleotide sequence encoding the H. tuberosuscytochrome p-450 enzyme.

SEQ ID NO:12 is the deduced amino acid sequence encoded by thenucleotide sequence encoding the H. tuberosus cytochrome p-450 enzyme.

SEQ ID NO:13 is the nucleotide sequence encoding the H. tuberosus p-450reductase enzyme.

SEQ ID NO:14 is the deduced amino acid sequence encoded by thenucleotide sequence encoding the H. tuberosus p-450 reductase enzyme.

DETAILED DESCRIPTION OF THE INVENTION

The present invention describes biological methods for the production ofPHCA. In one embodiment various bacteria and fungi were discovered thathave the ability to convert trans-cinnamate to PHCA. In anotherembodiment yeast PAL was transformed into a host E. coli and conversionof glucose to PHCA was demonstrated. In an alternate embodiment yeastPAL and the Jerusalem Artichoke plant cytochrome P-450 and thecytochrome P-450 reductase genes were incorporated into yeast hoststrain and the recombinant yeast had the ability to convert glucose toPHCA. In an additional embodiment a new bio-catalyst possessing enhancedtyrosine ammonia-lyase (TAL) activity was developed and the geneencoding this activity was used to transform a recombinant host for theproduction of PHCA. The evolution of TAL required isolation offunctional PAL gene, construction of a weak expression vector,mutagenesis and evolution of the PAL coding sequence, and selection ofvariants with improved TAL activity. The evolved TAL enzyme enablesmicroorganisms to produce PHCA from tyrosine in a single step.

The following abbreviations and definitions will be used for theinterpretation of the specification and the claims.

“Phenyl ammonia-lyase” is abbreviated PAL.

“Tyrosine ammonia-lyase” is abbreviated TAL.

“Para-hydroxycinnamic acid” is abbreviated PHCA.

“Cinnamate 4-hydroxylase” is abbreviated C4H.

As used herein the terms “cinnamic acid” and “cinnamate” are usedinterchangeably.

The term “TAL activity” refers to the ability of a protein to catalyzethe direct conversion of tyrosine to PHCA.

The term “PAL activity” refers to the ability of a protein to catalyzethe conversion of phenylalanine to cinnamic acid.

The term “P-450/P-450 reductase system” refers to a protein systemresponsible for the catalytic conversion of cinnamic acid to PHCA. TheP-450/P-450 reductase system is one of several enzymes or enzyme systemsknown in the art that perform a cinnamate 4-hydroxylase function. Asused herein the term “cinnamate 4-hydroxylase” will refer to the generalenzymatic activity that results in the conversion of cinnamic acid toPHCA, whereas the term “P-450/P-450 reductase system” will refer to aspecific binary protein system that has cinnamate 4-hydroxylaseactivity.

The term “PAL/TAL activity” or “PAL/TAL enzyme” refers to a proteinwhich contains both PAL and TAL activity. Such a protein has at leastsome specificity for both tyrosine and phenylalanine as an enzymaticsubstrate.

The term “mutant PAL/TAL” refers to a protein which has been derivedfrom a wild type PAL enzyme which has greater TAL activity than PALactivity. As such, a mutant PAL/TAL protein has a greater substratespecificity for tyrosine than for phenylalanine.

The term “catalytic efficiency” will be defined as the k_(cat)/K_(M) ofan enzyme. “Catalytic efficiency” will be used to quantitate thespecificity of an enzyme for a substrate.

The term “k_(cat)” is often called the “turnover number”. The term“k_(cat)” is defined as the maximum number of substrate moleculesconverted to products per active site per unit time, or the number oftimes the enzyme turns over per unit time. k_(cat)=Vmax/[E], where [E]is the enzyme concentration (Ferst In Enzyme Structure and Mechanism,2^(nd) ed.; W. H. Freeman: New York, 1985; pp 98-120).

The term “aromatic amino acid biosynthesis” means the biologicalprocesses and enzymatic pathways internal to a cell needed for theproduction of an aromatic amino acid.

The term “fermentable carbon substrate” refers to a carbon sourcecapable of being metabolized by host organisms of the present inventionand particularly carbon sources selected from the group consisting ofmonosaccharides, oligosaccharides, polysaccharides, and one-carbonsubstrates or mixtures thereof.

The term “complementary” is used to describe the relationship betweennucleotide bases that are capable to hybridizing to one another. Forexample, with respect to DNA, adenosine is complementary to thymine andcytosine is complementary to guanine. Accordingly, the instant inventionalso includes isolated nucleic acid fragments that are complementary tothe complete sequences as reported in the accompanying Sequence Listingas well as those substantially similar nucleic acid sequences.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” or “wild type gene” refers to a gene as found in naturewith its own regulatory sequences. “Chimeric gene” refers any gene thatis not a native gene, comprising regulatory and coding sequences thatare not found together in nature. Accordingly, a chimeric gene maycomprise regulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes.

“Coding sequence” refers to a DNA sequence that codes for a specificamino acid sequence.

“Suitable regulatory sequences” refer to nucleotide sequences locatedupstream (5′ non-coding sequences), within, or downstream (3′ non-codingsequences) of a coding sequence, and which influence the transcription,RNA processing or stability, or translation of the associated codingsequence. Regulatory sequences may include promoters, translation leadersequences, introns, and polyadenylation recognition sequences.

“Promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental conditions. Promoters whichcause a gene to be expressed in most cell types at most times arecommonly referred to as “constitutive promoters”. It is furtherrecognized that since in most cases the exact boundaries of regulatorysequences have not been completely defined, DNA fragments of differentlengths may have identical promoter activity.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of affecting the expression ofthat coding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment of the invention. Expression may also refer totranslation of mRNA into a polypeptide. “Antisense inhibition” refers tothe production of antisense RNA transcripts capable of suppressing theexpression of the target protein. “Overexpression” refers to theproduction of a gene product in transgenic organisms that exceeds levelsof production in normal or non-transformed organisms. “Co-suppression”refers to the production of sense RNA transcripts capable of suppressingthe expression of identical or substantially similar foreign orendogenous genes (U.S. Pat. No. 5,231,020).

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated into proteinby the cell. “cDNA” refers to a double-stranded DNA that iscomplementary to and derived from mRNA. “Sense” RNA refers to RNAtranscript that includes the mRNA and so can be translated into proteinby the cell. “Antisense RNA” refers to a RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target gene (U.S. Pat. No. 5,107,065).The complementarity of an antisense RNA may be with any part of thespecific gene transcript, i.e., at the 5′ non-coding sequence, 3′non-coding sequence, introns, or the coding sequence. “Functional RNA”refers to antisense RNA, ribozyme RNA, or other RNA that is nottranslated yet has an effect on cellular processes.

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” or “recombinant” or“transformed” organisms.

The terms “plasmid”, “vector” and “cassette” refer to an extrachromosomal element often carrying genes which are not part of thecentral metabolism of the cell, and usually in the form of circulardouble-stranded DNA molecules. Such elements may be autonomouslyreplicating sequences, genome integrating sequences, phage or nucleotidesequences, linear or circular, of a single- or double-stranded DNA orRNA, derived from any source, in which a number of nucleotide sequenceshave been joined or recombined into a unique construction which iscapable of introducing a promoter fragment and DNA sequence for aselected gene product along with appropriate 3′ untranslated sequenceinto a cell. “Transformation cassette” refers to a specific vectorcontaining a foreign gene and having elements in addition to the foreigngene that facilitate transformation of a particular host cell.“Expression cassette” refers to a specific vector containing a foreigngene and having elements in addition to the foreign gene that allow forenhanced expression of that gene in a foreign host.

The term “Lineweaver-Burk plot refers a plot of enzyme kinetic data forthe purpose of evaluating the kinetic parameters, K_(M) and V_(max).

Standard recombinant DNA and molecular cloning techniques used here arewell known in the art and are described by Sambrook, J., Fritsch, E. F.and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2^(nd) ed.;Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y., 1989(hereinafter “Maniatis”); and by Silhavy, T. J., Bennan, M. L. andEnquist, L. W. Experiments with Gene Fusions; Cold Spring HarborLaboratory: Cold Spring Harbor, N.Y., 1984; and by Ausubel, F. M. etal., In Current Protocols in Molecular Biology, published by GreenePublishing and Wiley-Interscience, 1987.

The present invention describes biological methods for the production ofPHCA. The method makes use of genes encoding proteins having cinnamate4-hydroxylase activity (C4H), phenylalanine ammonium-lyase (PAL)activity or tyrosine ammonium lyase (TAL) activity. A cinnamatehydroxylase activity will convert cinnamate to PHCA. Within the contextof the present invention a P-450/P-450 reductase system performs thisC4H function. A PAL activity will convert phenylalanine to PHCA in thepresence of a P-450/P-450 reductase system. These activities are linkedaccording to the following scheme:

A TAL activity will convert tyrosine directly to PHCA with nointermediate step according to the following scheme:

In one embodiment the method utilizes recombinant microbial host cellsexpressing an activity comprising both PAL and TAL functionalities inthe same protein. In this embodiment the host cell lacks the P-450/P-450reductase system and produces PHCA via the TAL route.

In another embodiment, the method utilizes a recombinant host comprisinga gene encoding the PAL activity in the presence of the gene encodingthe P-450/P-450 reductase system.

In an alternate embodiment the invention describes a method for theproduction of PHCA from cinnamate by organisms selected for their C4Hactivity.

The invention is useful for the biological production of PHCA which maybe used as a monomer for production of Liquid Crystal Polymers (LCP).LCP's may be used in electronic connectors and telecommunication andaerospace applications. LCP resistance to sterilizing radiation has alsoenabled these materials to be used in medical devices as well aschemical, and food packaging applications.

Genes:

The key enzymatic activities used in the present invention are encodedby a number of genes known in the art. The principal enzymes includecinnamate-4-hydroxylase (C4H) activity (P-450/P-450 reductase),phenylalanine ammonium lyase (PAL) and tyrosine ammonium lyase (TAL).

Phenylalanine Ammonium Lyase (PAL), Tyrosine Ammonium Lyase (TAL)Activities and the P-450/P-450 Reductase System:

Genes encoding PAL are known in the art and several have been sequencedfrom both plant and microbial sources (see for example EP 321488 [R.toruloides]; WO 9811205 [Eucalyptus grandis and Pinus radiata]; WO9732023 [Petunia]; JP 05153978 [Pisum sativum]; WO 9307279 [potato,rice]). The sequence of PAL genes is available (see for example GenBankAJ010143 and X75967). Where expression of a wild type PAL gene in arecombinant host is desired the wild type gene may be obtained from anysource including but not limited to, yeasts such as Rhodotorula sp.,Rhodosporidium sp. and Sporobolomyces sp.; bacterial organisms such asStreptomyces; and plants such as pea, potato, rice, eucalyptus, pine,corn, petunia, arabidopsis, tobacco, and parsley.

There are no known genes which encode an enzyme having exclusively TALactivity, i.e., which will use only tyrosine as a substrate for theproduction of PHCA. Several of the PAL enzymes mentioned above have somesubstrate affinity for tyrosine. Thus genes encoding TAL activity may beidentified and isolated concurrently with the PAL genes described above.For example, the PAL enzyme isolated from parsley (Appert et al., Eur.J. Biochem. 225:491 (1994)) and corn ((Havir et al., Plant Physiol.48:130 (1971)) both demonstrate the ability to use tyrosine as asubstrate. Similarly, the PAL enzyme isolated from Rhodosporidium(Hodgins D S, J. Biol. Chem. 246:2977 (1971)) also may use tyrosine as asubstrate. Such enzymes will be referred to herein as PAL/TAL enzymes oractivities. Where it is desired to create a recombinant organismexpressing a wild type gene encoding PAL/TAL activity, genes isolatedfrom maize, wheat, parsley, Rhizoctonia solani, Rhodosporidium,Sporobolomyces pararoseus and Rhodosporidium may be used as discussed inHanson and Havir, The Biochemistry of Plants; Academic: New York, 1981;Vol. 7, pp 577-625, where the genes from Rhodosporidium are preferred.

The invention provides a P-450/P-450 reductase system having C4Hactivity that is useful for the conversion of cinnamate to PHCA. Thissystem is well known in the art and has been isolated from a variety ofplant tissues. For example, the reductase as been isolated fromJerusalem Artichoke (Helianthus tuberosus), [embl locus HTU2NFR,accession Z26250.1]; parsley, (Petroselinum crispum) [Koopmann et al.,Proc. Natl. Acad. Sci. USA. 94 (26), 14954-14959 (1997), [locus AF024634accession AF024634.1]; California poppy (Eschscholzia californica),Rosco et al., Arch. Biochem. Biophys. 348 (2), 369-377 (1997), [locusECU67186 accession U67186.1]; Arabidopsis thaliana, [pir: locus S21531]; spring vetch (Vicia sativa), [pir: locus S37159]; mung bean, (Vignaradiata), Shet et al., Proc. Natl. Acad. Sci. U.S.A. 90 (7), 2890-2894(1993), [pir: locus A47298]; and opium poppy (Papaver somniferum),[locus PSU67185 accession U67185.1].

The cytochrome has been isolated from the Jerusalem Artichoke(Helianthus tuberosus),[embl locus HTTC4MMR, accession Z17369.1]; Zinniaelegans, [swissprot: locus TCMO_ZINEL, accession Q43240] Catharanthusroseus [swissprot: locus TCMO_CATRO, accession P48522]; Populustremuloides [swissprot: locus TCMO_POPTM, accession 024312]; Populuskitakamiensis [swissprot: locus TCMO_POPKI, accession Q43054];Glycyrrhiza echinata [swissprot: locus TCMO_GLYEC, accession Q96423];Glycine max [swissprot: locus TCMO_SOYBN, accession Q42797] as well asother sources.

Preferred in the instant invention are the genes encoding theP-450/P-450 reductase system isolated from Jerusalem Artichoke(Helianthus tuberosus) as set forth in SEQ ID NO:11 and SEQ ID NO:13.The skilled person will recognize that, for the purposes of the presentinvention, any cytochrome P-450/P-450 reductase system isolated from aplant will be suitable. As the sequence of the cytochrome gene (SEQ IDNO:11) ranges from about 92% identity (Zinnia elegans, Q43240) to about63% identity (Phaseolus vulgaris, embl locus PV09449, accessionY09449.1) to known P-450 cytochromes in these systems, it iscontemplated that any P-450 cytochrome isolated from a plant having atleast 63% identity to SEQ ID NO:11 will be suitable in the presentinvention. Similarly, as the p-450 reductase in the system (SEQ IDNO:13) ranges from about 79% identity (parsley, AF024634.1] to about 68%identity (opium poppy, U67185.1) identity to known reductases P-450's itis contemplated that any P-450 reductase isolated from a plant having atleast 68% identity to SEQ ID NO:13 will be suitable in the presentinvention.

Methods of obtaining these or homologous wild type genes usingsequence-dependent protocols are well known in the art. Examples ofsequence-dependent protocols include, but are not limited to, methods ofnucleic acid hybridization, and methods of DNA and RNA amplification asexemplified by various uses of nucleic acid amplification technologies(e.g., polymerase chain reaction (PCR), ligase chain reaction (LCR)).

For example, genes encoding homologs or anyone of the mentionedactivites (PAL, TAL or the P-450/P-450 reductase system) could beisolated directly by using all or a portion of the known sequences asDNA hybridization probes to screen libraries from any desired plant,fungi, yeast, or bacteria using methodology well known to those skilledin the art. Specific oligonucleotide probes based upon the literaturenucleic acid sequences can be designed and synthesized by methods knownin the art (Maniatis, supra). Moreover, the entire sequences can be useddirectly to synthesize DNA probes by methods known to the skilledartisan such as random primers DNA labeling, nick translation, orend-labeling techniques, or RNA probes using available in vitrotranscription systems. In addition, specific primers can be designed andused to amplify a part of or full-length of the instant sequences. Theresulting amplification products can be labeled directly duringamplification reactions or labeled after amplification reactions, andused as probes to isolate full length cDNA or genomic fragments underconditions of appropriate stringency.

In addition, two short segments of the literature sequences may be usedin polymerase chain reaction protocols to amplify longer nucleic acidfragments encoding homologous genes from DNA or RNA. The polymerasechain reaction may also be performed on a library of cloned nucleic acidfragments wherein the sequence of one primer is derived from theliterature sequences, and the sequence of the other primer takesadvantage of the presence of the polyadenylic acid tracts to the 3′ endof the mRNA precursor encoding bacterial genes. Alternatively, thesecond primer sequence may be based upon sequences derived from thecloning vector. For example, the skilled artisan can follow the RACEprotocol (Frohman et al., PNAS USA 85:8998 (1988)) to generate cDNAs byusing PCR to amplify copies of the region between a single point in thetranscript and the 3′ or 5′ end. Primers oriented in the 3′ and 5′directions can be designed from the literature sequences. Usingcommercially available 3′ RACE or 5′ RACE systems (BRL), specific 3′ or5′ cDNA fragments can be isolated (Ohara et al., PNAS USA 86:5673(1989); Loh et al., Science 243:217 (1989)).

Mutant PAL/TAL Activities:

It is an object of the present invention to provide a mutant PAL/TALactivity having a greater substrate specificity for tyrosine than forphenylalanine. Typically the approach will involve the selection of anorganism having a PAL/TAL activity with a higher substrate specificityfor tyrosine than for phenylalanine. Generally, the substratespecificity is quantitated by k_(cat)/K_(M) (catalytic efficiency),calculated on the basis of the number of active sites identified in theenzyme.

Phenylalanine ammonia-lyase has a molecular weight of about 330,000 andconsists of four identical subunits of about 80 KD (Havir et al.,Biochemistry 14:1620-1626 (1975)). It has been suggested that PALcontains a catalytically essential dehydroalanine residue (Hanson etal., Arch. Biochem. Biophys. 141:1-17 (1970)). Ser-202 of PAL fromparsley has been indicated as the precursor of the dehydroalanine(Langer et al., Biochemistry, 36:10867-10871 (1997)). The k_(cat) forPAL was calculated using information available from recent studies onthe crystal structure of a homologous enzyme, histidine ammonia-lyase(HAL). These studies have revealed that the reactive electrophilicresidue in the active site of the enzyme is a4-methylidene-ididazole-5-one, which is autocatalytically formed bycyclization and dehydration of residues 142-144 containing theAla-Ser-Gly sequence (Schwede et al., Biochemistry 38:5355-5361 (1999)).Since all tetrameric PAL enzymes studied so far, also contain theAla-Ser-Gly sequence at each of their active sites, it is likely thateach active site of PAL also contains a 4-methylidene-ididazole-5-oneformed from this sequence.

Within the context of the present invention, the suitable wildtypeenzyme selected for mutagenesis has a catalytic efficiency of about4.14×10³ to 1×10⁹ M⁻¹sec⁻¹ for tyrosine where a catalytic efficiency ina range of about of about 1×10⁴ M⁻¹sec⁻¹ to about 5×10⁴ M⁻¹sec⁻¹ ispreferred.

The process of the selection of a suitable PAL/TAL enzyme, involvesconstruction of a weak expression vector, mutagenesis and evolution ofthe PAL coding sequence, and finally selection of variants with improvedTAL activity.

Mutagenesis of PAL:

A variety of approaches may be used for the mutagenesis of the PAL/TALenzyme. Two suitable approaches used herein include error-prone PCR(Leung et al., Techniques, 1:11-15 (1989) and Zhou et al., Nucleic AcidsRes. 19:6052-6052 (1991) and Spee et al., Nucleic Acids Res. 21:777-778(1993)) and in vivo mutagenesis.

The principal advantage of error-prone PCR is that all mutationsintroduced by this method will be within the PAL gene, and any changemay be easily controlled by changing the PCR conditions. Alternativelyin vivo mutagenesis, may be employed using commercially availablematerials such as E. coli XL1-Red strain, and the Epicurian coli XL1-Redmutator strain from Stratagene (Stratagene, La Jolla, Calif., Greenerand Callahan, Strategies 7:32-34 (1994)). This strain is deficient inthree of the primary DNA repair pathways (mutS, mutD and mutT),resulting in a mutation rate 5000-fold higher than that of wild-type. Invivo mutagenesis does not depend on ligation efficiency (as witherror-prone PCR), however a mutation may occur at any region of thevector and the mutation rates are generally much lower.

Alternatively, it is contemplated that a mutant PAL/TAL enzyme withenhanced TAL activity may be constructed using the method of “geneshuffling” (U.S. Pat. No. 5,605,793; U.S. Pat. No. 5,811,238; U.S. Pat.No. 5,830,721; and U.S. Pat. No. 5,837,458). The method of geneshuffling is particularly attractive due to its facile implementation,and high rate of mutagenesis. The process of gene shuffling involves therestriction of a gene of interest into fragments of specific size in thepresence of additional populations of DNA regions of both similarity toor difference to the gene of interest. This pool of fragments will thendenature and then reanneal to create a mutated gene. The mutated gene isthen screened for altered activity.

Wild type PAL/TAL sequences may be mutated and screened for altered orenhanced TAL activity by this method. The sequences should be doublestranded and can be of various lengths ranging from 50 bp to 10 kb. Thesequences may be randomly digested into fragments ranging from about 10bp to 1000 bp, using restriction endonucleases well known in the art(Maniatis supra). In addition to the full length sequences, populationsof fragments that are hybridizable to all or portions of the sequencemay be added. Similarly, a population of fragments which are nothybridizable to the wild type sequence may also be added. Typicallythese additional fragment populations are added in about a 10 to 20 foldexcess by weight as compared to the total nucleic acid. Generally thisprocess will allow generation of about 100 to 1000 different specificnucleic acid fragments in the mixture. The mixed population of randomnucleic acid fragments are denatured to form single-stranded nucleicacid fragments and then reannealed. Only those single-stranded nucleicacid fragments having regions of homology with other single-strandednucleic acid fragments will reanneal. The random nucleic acid fragmentsmay be denatured by heating. One skilled in the art could determine theconditions necessary to completely denature the double stranded nucleicacid. Preferably the temperature is from 80° C. to 100° C. The nucleicacid fragments may be reannealed by cooling. Preferably the temperatureis from 20° C. to 75° C. Renaturation can be accelerated by the additionof polyethylene glycol (“PEG”) or salt. The salt concentration ispreferably from 0 mM to 200 mM. The annealed nucleic acid fragments arenext incubated in the presence of a nucleic acid polymerase and dNTP's(i.e., dATP, dCTP, dGTP and dTTP). The nucleic acid polymerase may bethe Klenow fragment, the Taq polymerase or any other DNA polymeraseknown in the art. The polymerase may be added to the random nucleic acidfragments prior to annealing, simultaneously with annealing or afterannealing. The cycle of denaturation, renaturation and incubation in thepresence of polymerase is repeated for a desired number of times.Preferably the cycle is repeated from 2 to 50 times, more preferably thesequence is repeated from 10 to 40 times. The resulting nucleic acid isa larger double-stranded polynucleotide of from about 50 bp to about 100kb and may be screened for expression and altered TAL activity bystandard cloning and expression protocols. (Maniatis supra).

Irrespective of the method of mutagenesis it is contemplated that a genemay be evolved having a catalytic efficiency of about 4.14×10³ M⁻¹sec⁻¹to about 1×10⁹ M⁻¹sec⁻¹ where an catalytic efficiency of about 12.6×10³M⁻¹sec⁻¹ is typical.

Selection of Variants with Improved TAL Activity

In order to select for those mutants having genes encoding proteins withenhanced TAL activity, a selection system based on the reversibility ofthe tyrosine to PHCA reaction was developed. It will be appreciated thatthe TAL activity responsible for the conversion of tyrosine to PHCA isin a state of equilibrium with the opposite reaction. Mutant genes werecloned by standard methods into E. coli tyrosine auxotrophs, unable togrow in the absence of tyrosine. Transformants were plated on tyrosineminus medium in the presence of suitable concentrations of PHCA. Thosecolonies which grew under these conditions were picked and analyzed forthe presence of the mutant gene. In this fashion, a gene was isolatedthat had a catalytic efficiency of about 12.6×10³ M⁻¹sec⁻¹ and a ratioof TAL catalytic activity to PAL catalytic activity of 1.7 compared to0.5 for the wild type.

The skilled person will be able to envision additional screens for theselection of genes encoding enhanced TAL activity. For example, it iswell known that Acinetobacter calcoaceticus DSM 586 (ATCC 33304) is ableto efficiently degrade p-coumaric acid (PHCA) and use it as a solecarbon source (Delneri et al., Biochim. Biophys. Acta 1244:363-367(1995)). The proposed pathway for this degradation is shown as PathwayI;

Pathway I

p-hydroxycinnamic acid→p-hydroxybenzoic acid→protocatechuic acid

The enzymes involved in this proposed pathway are all induced by theaddition of PHCA to cell cultures. By transformation of a TAL gene intoA. calcoaceticus (ATCC 3304), or into other microorganisms able to usePHCA as a sole carbon source, the above pathway is now modified to showtyrosine as a substrate for PHCA, as illustrated in Pathway II;

Pathway II

L-tyrosine→p-hydroxycinnamic acid→p-hydroxybenzoic acid→protocatechuicacid

It will be appreciated that cells possessing the elements of Pathway II,when grown on PHCA will show more vigorous growth than those possessingonly Pathway I. Thus, this system may be used as a screen for theidentification of genes possessing TAL activity. This selection systemhas the added advantage of avoiding the effects of inhibitory levels ofPHCA as the cell contains a pathway to degrade this compound furtheruntil the carbon enters central metabolism.

Production Organisms:

Microbial Hosts

The production organisms of the present invention will include anyorganism capable of expressing the genes required for the PHCAproduction. Typically the production organism will be restricted tomicroorganisms and plants.

Microorganisms useful in the present invention for the production ofPHCA may include, but are not limited to bacteria, such as the entericbacteria (Escherichia, and Salmonella for example) as well as Bacillus,Acinetobacter, Streptomyces, Methylobacter, Rhodococcus and Pseudomona;Cyanobacteria, such as Rhodobacter and Synechocystis; yeasts, such asSaccharomyces, Zygosaccharomyces, Kluyveromyces, Candida, Hansenula,Debaryomyces, Mucor, Pichia and Torulopsis; and filamentous fungi suchas Aspergillus and Arthrobotrys, and algae for example. The PAL, PAL/TALand the P-450 and P-450 reductase genes of the present invention may beproduced in these and other microbial hosts to prepare large,commercially useful amounts of PHCA.

Microbial expression systems and expression vectors containingregulatory sequences that direct high level expression of foreignproteins are well known to those skilled in the art. Any of these couldbe used to construct chimeric genes for production of PHCA. Thesechimeric genes could then be introduced into appropriate microorganismsvia transformation to allow for expression of high level of the enzymes.

Vectors or cassettes useful for the transformation of suitable microbialhost cells are well known in the art. Typically the vector or cassettecontains sequences directing transcription and translation of therelevant gene, a selectable marker, and sequences allowing autonomousreplication or chromosomal integration. Suitable vectors comprise aregion 5′ of the gene which harbors transcriptional initiation controlsand a region 3′ of the DNA fragment which controls transcriptionaltermination. It is most preferred when both control regions are derivedfrom genes homologous to the transformed host cell, although it is to beunderstood that such control regions need not be derived from the genesnative to the specific species chosen as a production host.

Initiation control regions or promoters, which are useful to driveexpression of the relevant genes in the desired host cell are numerousand familiar to those skilled in the art. Virtually any promoter capableof driving these genes is suitable for the present invention includingbut not limited to CYC1, HIS3, GAL1, GAL10, ADH1, PGK, PHO5, GAPDH,ADC1, TRP1, URA3, LEU2, ENO, TPI (useful for expression inSaccharomyces); AOX1 (useful for expression in Pichia); and lac, trp,lP_(L), lP_(R), T7, tac, and trc (useful for expression in Escherichiacoli).

Termination control regions may also be derived from various genesnative to the preferred hosts. Optionally, a termination site may beunnecessary, however, it is most preferred if included.

Where commercial production of PHCA is desired a variety of fermentationmethodologies may be applied. For example, large scale production may beeffected by both batch or continuous fermentation.

A classical batch fermentation is a closed system where the compositionof the media is set at the beginning of the fermentation and not subjectto artificial alterations during the fermentation. Thus, at thebeginning of the fermentation the medium is inoculated with the desiredmicroorganism or microorganisms and fermentation is permitted to occuradding nothing to the system. Typically, however, the concentration ofthe carbon source in a “batch” fermentation is limited and attempts areoften made at controlling factors such as pH and oxygen concentration.In batch systems the metabolite and biomass compositions of the systemchange constantly up to the time the fermentation is stopped. Withinbatch cultures cells moderate through a static lag phase to a highgrowth log phase and finally to a stationary phase where growth rate isdiminished or halted. If untreated, cells in the stationary phase willeventually die. Cells in the log phase generally are responsible for thebulk of production of end product or intermediate.

A variation on the standard batch system is the Fed-Batch system.Fed-Batch fermentation processes are also suitable in the presentinvention and comprise a typical batch system with the exception thatthe substrate is added in increments as the fermentation progresses.Fed-Batch systems are useful when catabolite repression is apt toinhibit the metabolism of the cells and where it is desirable to havelimited amounts of substrate in the medium. Measurement of the actualsubstrate concentration in Fed-Batch systems is difficult and istherefore estimated on the basis of the changes of measurable factorssuch as pH, dissolved oxygen and the partial pressure of waste gasessuch as CO₂. Batch and Fed-Batch fermentations are common and well knownin the art and examples may be found in Brock, T. D.; Biotechnology: ATextbook of Industrial Microbiology, 2nd ed.; Sinauer Associates:Sunderland, Mass., 1989; or Deshpande, M. V. Appl. Biochem. Biotechnol.36:227, (1992), herein incorporated by reference.

Commercial production of PHCA may also be accomplished with continuousfermentation. Continuous fermentation is an open system where a definedfermentation medium is added continuously to a bioreactor and an equalamount of conditioned medium is removed simultaneously for processing.Continuous fermentation generally maintains the cultures at a constanthigh density where cells are primarily in their log phase of growth.

Continuous fermentation allows for modulation of any number of factorsthat affect cell growth or end product concentration. For example, onemethod will maintain a limiting nutrient such as the carbon source ornitrogen level at a fixed rate and allow all other parameters tomoderate. In other systems a number of factors affecting growth can bealtered continuously while the cell concentration, measured by themedium turbidity, is kept constant. Continuous systems strive tomaintain steady state growth conditions and thus the cell loss due tothe medium removal must be balanced against the cell growth rate in thefermentation. Methods of modulating nutrients and growth factors forcontinuous fermentation processes as well as techniques for maximizingthe rate of product formation are well known in the art of industrialmicrobiology and a variety of methods are detailed by Brock, supra.

For production of PHCA via the PAL route in the presence of theP-450/P-450 reductase system any medium that will support the growth ofthe cells is suitable. Where, however, production of PHCA is desired aspart of the natural carbon flow of the microorganism, the fermentationmedium must contain suitable carbon substrates. Suitable substrates mayinclude but are not limited to monosaccharides such as glucose,raffinose and fructose, oligosaccharides such as lactose or sucrose,polysaccharides such as starch or cellulose or mixtures thereof andunpurified mixtures from renewable feedstocks such as cheese wheypermeate, cornsteep liquor, sugar beet molasses, and barley malt.Additionally the carbon substrate may also be one-carbon substrates suchas carbon dioxide, formaldehyde, formate or methanol for which metabolicconversion into key biochemical intermediates has been demonstrated.

Plant Hosts

Alternatively, the present invention provides for the production of PHCAin plant cells harboring the relevant PAL, PAL/TAL and the P-450 andP-450 reductase genes. Preferred plant hosts will be any variety thatwill support a high production level of PHCA or PHCA-glucosideconjugate. Suitable green plants will include but are not limited tosoybean, rapeseed (Brassica napus, B. campestris), sunflower (Helianthusannus), Jerusalem artichoke (Helianthus tuberosis), cotton (Gossypiumhirsutum), corn, tobacco (Nicotiana tabacum), alfalfa (Medicago sativa),wheat (Triticum sp), barley (Hordeum vulgare), oats (Avena sativa, L),sorghum (Sorghum bicolor), rice (Oryza sativa), Arabidopsis, cruciferousvegetables (broccoli, cauliflower, cabbage, parsnips, etc.), melons,carrots, celery, parsley, tomatoes, potatoes, strawberries, peanuts,grapes, grass seed crops, sugar beets, sugar cane, beans, peas, rye,flax, hardwood trees, softwood trees, and forage grasses. Overexpressionof the necessary genes of the present invention may be accomplished byfirst constructing chimeric genes in which the coding regions areoperably linked to promoters capable of directing expression of a genein the desired tissues at the desired stage of development. For reasonsof convenience, the chimeric genes may comprise promoter sequences andtranslation leader sequences derived from the same genes. 3′ Non-codingsequences encoding transcription termination signals must also beprovided. The instant chimeric genes may also comprise one or moreintrons in order to facilitate gene expression.

Any combination of any promoter and any terminator capable of inducingexpression of a coding region may be used in the chimeric geneticsequence. Some suitable examples of promoters and terminators includethose from nopaline synthase (nos), octopine synthase (ocs) andcauliflower mosaic virus (CaMV) genes. One type of efficient plantpromoter that may be used is a high level plant promoter. Suchpromoters, in operable linkage with the genetic sequences of the presentinvention should be capable of promoting expression of the present geneproduct. High level plant promoters that may be used in this inventioninclude the promoter of the small subunit (ss) of theribulose-1,5-bisphosphate carboxylase for example from soybean(Berry-Lowe et al., J. Molecular and App. Gen. 1:483-498 (1982)), andthe promoter of the chlorophyll a/b binding protein. These two promotersare known to be light-induced in plant cells (see for example, Cashmore,A. Genetic Engineering of Plants, an Agricultural Perspective; Plenum:New York, 1983; pp 29-38; Coruzzi et al., J. Biol. Chem. 258:1399(1983), and Dunsmuir et al., J. Mol. Appl. Genetics 2:285 (1983)).

Plasmid vectors comprising the instant chimeric genes can thenconstructed. The choice of plasmid vector depends upon the method thatwill be used to transform host plants. The skilled artisan is well awareof the genetic elements that must be present on the plasmid vector inorder to successfully transform, select and propagate host cellscontaining the chimeric gene. The skilled artisan will also recognizethat different independent transformation events will result indifferent levels and patterns of expression (Jones et al., EMBO J.4:2411-2418 (1985); De Almeida et al., Mol. Gen. Genetics 218:78-86(1989)), and thus that multiple events must be screened in order toobtain lines displaying the desired expression level and pattern. Suchscreening may be accomplished by Southern analysis of DNA blots(Southern et al., J. Mol. Biol. 98:503 (1975)), Northern analysis ofmRNA expression (Kroczek, J. Chromatogr. Biomed. Appl.., 618:133-145(1993), Western analysis of protein expression, enzymatic activityanalysis of expressed gene product, or phenotypic analysis.

For some applications it will be useful to direct the gene products ofthe PHCA producing genes to different cellular compartments. It is thusenvisioned that the chimeric genes described above may be furthersupplemented by altering the coding sequences to encode enzymes withappropriate intracellular targeting sequences such as transit sequences(Keegstra, K., Cell 56:247-253 (1989)), signal sequences or sequencesencoding endoplasmic reticulum localization (Chrispeels, J. J., Ann.Rev. Plant Phys. Plant Mol. Biol. 42:21-53 (1991)), or nuclearlocalization signals (Raikhel, N., Plant Phys. 100:1627-1632 (1992))added and/or with targeting sequences that are already present removed.While the references cited give examples of each of these, the list isnot exhaustive and more targeting signals of utility may be discoveredin the future that are useful in the invention.

Optionally it is contemplated that PHCA production in plants may beenhanced by the antisense inhibition or co-suppression of genes encodingenzymes down stream of PHCA. These enzymes may serve to transform PHCAinto less useful products and prevent PHCA accumulation. Transgenicplants comprising constructs harboring genes encoding these down streamgenes in antisense conformation may be useful in enhancing PHCAaccumulation. Similarly, the same genes, overexpressed may serve toenhance PHCA accumulation by gene co-suppression. Thus, the skilledperson will appreciate that chimeric genes designed to express antisenseRNA (U.S. Pat. No. 5,107,065) for all or part of the instant down streamgenes can be constructed by linking the genes or gene fragment inreverse orientation to plant promoter sequences. Either theco-suppression or antisense chimeric genes could be introduced intoplants via transformation whereby expression of the correspondingendogenous genes are reduced or eliminated.

Methods of Production:

The present invention provides several methods for the bio-production ofPHCA. In one embodiment cinnamate may be contacted with an organismwhich contains the requisite C4H activity. These organisms may be wildtype or recombinant. Several organisms were uncovered by the presentinvention as having the ability to convert cinnamate to PCHA includingStreptomyces griseus (ATCC 13273, ATCC 13968, TU6), Rhodococcuserythropolis (ATCC 4277), Aspergillus petrakii (ATCC 12337), Aspergillusniger (ATCC 10549) and Arthrobotrys robusta (ATCC 11856).

In an alternate embodiment, yeast PAL and the plant cytochrome P-450 andthe cytochrome P-450 reductase genes were incorporated into yeast hoststrains and the recombinant yeast demonstrated the ability to convertglucose to PHCA. Saccharomyces cerevisiae was chosen for this means ofproduction, however it will be appreciated by the skilled artisan that avariety of yeasts will be suitable, including, but not limited to thosemicrobial production organisms described above. Similarly, glucose wasemployed as a carbon substrate, however a variety of other fermentablecarbon substrates may be used.

In a preferred embodiment PHCA may be produced from a recombinantmicroorganism or plant cell which lacks a P-450/P-450 reductase systemand harbors a PAL/TAL enzyme where the enzyme has a minimum level of TALactivity and the carbon flow is directed from a fermentable carbonsource through tyrosine to PHCA.

The present invention is further defined in the following Examples. Itshould be understood that these Examples, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and these Examples, one skilled in the art canascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usage andconditions.

EXAMPLES

General Methods:

Procedures required for PCR amplification, DNA modifications by endo-and exonucleases for generating desired ends for cloning of DNA,ligation, and bacterial transformation are well known in the art.Standard molecular cloning techniques used here are well known in theart and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T.Molecular Cloning: A Laboratory Manual, 2^(nd) ed.; Cold Spring HarborLaboratory: Cold Spring Harbor, N.Y., 1989 (hereinafter “Maniatis”); andby Silhavy, T. J., Bennan, M. L. and Enquist, L. W. Experiments withGene Fusions; Cold Spring Harbor Laboratory: Cold Spring, N.Y., 1984 andby Ausubel et al., Current Protocols in Molecular Biology; GreenePublishing and Wiley-Interscience; 1987.

Materials and methods suitable for the maintenance and growth ofbacterial cultures are well known in the art. Techniques suitable foruse in the following examples may be found as set out in Manual ofMethods for General Bacteriology; Phillipp Gerhardt, R. G. E. Murray,Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg andG. Briggs Phillips, Eds., American Society for Microbiology: Washington,DC, 1994 or by Brock, T. D.; Biotechnology: A Textbook of IndustrialMicrobiology, 2nd ed.; Sinauer Associates: Sunderland, Mass., 1989. Allreagents, restriction enzymes and materials used for the growth andmaintenance of bacterial cells were obtained from Aldrich Chemicals(Milwaukee, Wis.), DIFCO Laboratories (Detroit, Mich.), GIBCO/BRL(Gaithersburg, Md.), or Sigma Chemical Company (St. Louis, Mo.) unlessotherwise specified.

PCR reactions were run on GeneAMP PCR System 9700 using Amplitaq orAmplitaq Gold enzymes (PE Applied Biosystems, Foster City, Calif.). Thecycling conditions and reactions were standardized according to themanufactures instructions.

The meaning of abbreviations is as follows: “sec” means second(s),“min”means minute(s), “h” means hour(s), “d” means day(s), “μL” meansmicroliter, “mL” means milliliters, “L” means liters, “mm” meansmillimeters, “nm” means nanometers, “mM” means millimolar, “M” meansmolar, “mmol” means millimole(s), “μmole” mean micromole”, “g” meansgram, “μg” means microgram and “ng” means nanogram, “U” means units, and“mU” means milliunits.

Strains, vectors and culture conditions:

Tyrosine auxotrophic Escherichia coli strain AT2471 and wild typeEscherichia coli W3110 were originally obtained from Coli Genetic StockCenter (CGSC #4510), Yale University, New Haven, Conn.). Epicurian coliXL1-Red strain was purchased from Stratagene. Escherichia coli BL21(DE3)cells were used for enzyme over-expression (Shuster, B. and Retey, J.,FEBS Lett. 349:252-254 (1994)). pBR322 and pET-24d vectors werepurchased from New England Biolab (Bevely, Mass.) and Novagen (Madison,Wis.), respectively. pKK223-3 was purchased from Amersham Pharmacia.

Growth Media for Rhodosporidium toruloides:

Complex Medium: Rhodosporidium toruloides (ATCC number 10788) wascultured in a medium containing malt extract (1.0%), yeast extract(0.10%) and L-phenylalanine (0.10%) in deionized water. Difco certifiedBacto-malt and Bacto-yeast extract were used. A solution of malt andyeast extract was autoclaved without phenylalanine. An aliquot (50 mL)of a filter-sterilized 2% solution of phenylalanine was added to the 1.0L autoclaved malt and yeast extract solution. (Abell et al.,“Phenylalanine Ammonia-lyase from Yeast Rhodotorula glutinis”, MethodsEnzymol. 142:242-248 (1987)).

Minimal Medium: The medium contained 50 mM potassium phosphate buffer(pH 6.2), MgSO₄ (100 mg/L), biotin (10 mg/L) and L-phenylalanine (2.5g/L) in deionized water. A solution of phosphate buffer was autoclavedwithout the other ingredients. A solution of L-phenylalanine (25 g/L),MgSO₄ (1.0 g/L) and biotin (0.1 g/L) in 1.01 of 50 mM potassiumphosphate buffer (pH 6.2) was filter sterilized and 100 mL added to 900mL of the autoclaved phosphate buffer. Final concentrations of theingredients were: KH₂PO₄ (5.55 g/L); K₂HPO₄ (1.61 g/L) MgSO₄ (100 mg/L);biotin (10 mg/L) and L-phenylalanine (2.5 g/L) (Marusich, W. C., Jensen,R. A. and Zamir, L. O. “Induction of L-Phenylalanine Ammonia-LyaseDuring Utilization of Phenylalanine as a Carbon or Nitrogen Source inRhodosporidium toruloides”, J. Bacteriol. 146:1013-1019 (1981)).

Enzyme Activity Assay:

The PAL or TAL activity of the purified enzymes were measured using aspectrophotometer according to Abell et al., “PhenylalanineAmmonia-lyase from Yeast Rhodotorula glutinis,” Methods Enzymol.142:242-248 (1987). The spectrophotometric assay for PAL determinationwas initiated by the addition of the enzyme to a solution containing 1.0mM L-phenylalanine and 50 mM Tris-HCl (pH 8.5). The reaction was thenfollowed by monitoring the absorbance of the product, cinnamic acid, at290 nm using a molar extinction coefficient of 9000 cm⁻¹. The assay wasrun over a 5 min period using an amount of enzyme that producedabsorbance changes in the range of 0.0075 to 0.018/min. One unit ofactivity indicated deamination of 1.0 μmol of phenylalanine to cinnamicacid per minute. The TAL activity was similarly measured using tyrosinein the reaction solution. The absorbance of the para-hydroxycinnamicacid produced was followed at 315 nm and the activity was determinedusing an extinction coefficient of 10,000 cm⁻¹ for PHCA. One unit ofactivity indicated deamination of 1.0 μmol of tyrosine topara-hydroxycinnamic acid per minute.

SDS Gel Electrophoresis:

The 8-25% native PhastGels were run with 4.0 μg of protein per lane andstained with Coomassie blue. Pharmacia High Molecular Weight (HMW)markers and grade I PAL from Sigma were used as standards.

Sample Preparation for HPLC Analysis:

An HPLC assay was developed for measuring the levels of cinnamic acidand PHCA formed by the whole cells. In a typical assay, followingcentrifugation of a culture grown in the medium of choice, 20-1000 μL ofthe supernatant was acidified with phosphoric acid, filtered through a0.2 or 0.45 micron filter and analyzed by the HPLC to determine theconcentration of PHCA and cinnamic acid in the growth medium.Alternatively, following centrifugation, the cells were resuspended in100 mM Tris-HCl (pH 8.5) containing 1.0 mM tyrosine or 1.0 mMphenylalanine and incubated at room temperature for 1.0-16 h. A filteredaliquot (20-1000 μL) of this suspension was then analyzed.

The HPLC Method:

A Hewlett Packard 1090M HPLC system with an auto sampler and a diodearray UV/vis detector was used with a reverse-phase Zorbax SB-C8 column(4.6 mm×250 mm) supplied by MAC-MOD Analytical Inc. Flow rate of 1.0 mLper min, at column temperature of 40° C. was carried out. The UVdetector was set to monitor the eluant at 250, 230, 270, 290 and 310 nmwavelengths.

Solvents/Gradients: Solvent A Solvent B Time (min) Methanol 0.2% TFA 0.010% 90% 0.1 10% 90% 9.0 35% 65% 9.1 50% 50% 14.0 50% 50% 18.0  0%  0%21.0  0%  0%

Retention time (RT) of related metabolites using the HPLC systemdescribed above are summarized below.

Compounds (1.0 mM) RT (min)  1. tyrosine 6.7  2. phenylalanine 9.4  3.4-hydroxybenzoic acid (PHBA) 11.6  4. 3,4-dihydroxycinnamate (caffeicacid) 12.5  5. 3-(4-hydroxyphenyl)propionate 13.3  6.4-hydroxyphenylpyruvate 13.6  7. 4-hydroxyacetaphenone 14.0  8.4-hydroxycinnamic acid (PHCA) 14.2  9. 2-hydroxycinnamic acid (OHCA)15.3 10. benzoic acid 15.5 11. coumarin 16.0 12. cinnamyl alcohol 17.313. phenylpyruvate 18.1 14. cinnamic acid 18.3

MONO Q buffer:

The buffer used for these analyses was a 50 mM potassium phosphate, pH7.0, as the starting buffer followed by a 400 mM potassium phosphatebuffer, pH 7.2 as eluent for the Mono-Q column.

Example 1

Microorganisms for Conversion of Cinnamic Acid to PHCA

Example 1 describes screening of various microorganisms for the presenceof cinnamate hydroxylases and investigation of their ability to convertcinnamic acid to PHCA.

In order to discover microorganisms with cinnamate hydroxylase activity,over 150 different strains of bacteria and fungi were screened for theirability to convert cinnamic acid to PHCA. A two-stage fermentationprotocol was used. Microorganisms were first grown in the medium forthree days and then a 20% inoculum was used to start the second stagecultures. Following 24 h growth in stage two, cinnamic acid was added,samples were taken at intervals and analyzed by HPLC for the presence ofPHCA.

Growth Media:

ATCC Medium # 196—Yeast/malt Medium

This medium contained (in grams per liter): malt extract, 6.0; maltose,1.8; dextrose, 6.0; and yeast extract, 1.2. The pH was adjusted to 7.0.

ATCC Medium # 5—Sporulation Broth

This medium contained (in grams per liter): yeast extract, 1.0; beefextract, 1.0; tryptone, 2.0; and glucose, 10.0.

Soybean Flour/glycerol Medium (SBG):

This medium contained (in grams per liter): glycerol, 20; yeast extract,5.0; soybean flour, 5.0; sodium chloride, 5.0; potassium phosphatedibasic, 5.0. The pH was adjusted to 7.0.

Potato-dextrose/yeast Medium (PDY):

This medium which contained (in grams per liter): potato dextrose broth,24.0; yeast extract, 5.0; was used for growth of fungal strains.

Of the 100-150 microorganisms tested, three separate strains ofStreptomyces griseus (ATCC 13273, ATCC 13968, TU6), the bacteriumRhodococcus erythropolis (ATCC 4277), and the fungal strains,Aspergillus petrakii (ATCC 12337), Aspergillus niger (ATCC 10549) andArthrobotrys robusta (ATCC 11856) demonstrated the ability to convertcinnamic acid to PHCA. The results indicated that Streptomycetes, ingeneral, and Streptomyces griseus, in particular, appeared to be mostactive in this hydroxylation. Further studies were therefore performedusing the following strains of Streptomyces griseus (ATCC 13273, ATCC13968, TU6).

The ability of the Streptomyces griseus strains to para-hydroxylatecinnamic acid to PHCA while growing in three complex media (SBG,sporulation broth and yeast/malt media) was examined. The two stagefermentation protocol with SBG, sporulation broth and malt/yeast mediawas used. Samples were taken at various time intervals and analyzed byHPLC for the presence of PHCA. Data is shown below in Table 1.

TABLE 1 Effect of Different Media on the Ability of Various Strains ofStreptomyces griseus to Convert Cinnamic Acid to PHCA PHCA Production(μM) malt/yeast SBG sporulation broth Strain 13273:  4 h 0.93 116.812.75 18 h 0 360.75 14.31 24 h 5.36 407.27 12.14 42 h 0 350.08 7.26 60 h0 363.94 9.79 Strain TU6:  4 h 0 2.54 0.62 18 h 0 20.76 0.64 24 h 1.2322.23 0.54 42 h 0.93 30.46 0.95 60 h 1.24 50.82 1.84 Strain 13968:  4 h0 2.92 41.82 18 h 0 6.02 267.38 24 h 0 20.55 282.29 42 h 0 127.25 177.4460 h 0 172.78 160.71

As is seen by the data, among Streptomyces griseus strains tested, ATCC13273 followed by ATCC 13968 and TU6 were the most active in producingPHCA when grown in the SBG medium. With ATCC 13968 strain ofStreptomyces griseus growth in both SBG and sporulation broth resultedin the ability to convert cinnamic acid to PHCA. Cells (ATCC 13968)grown on sporulation medium showed the highest ability to produce PHCAafter 24 h of growth while those grown on SGB medium reached theirmaximum PHCA producing activity after 60 h.

Example 2

Screening of Microorganisms Containing Optimal TAL/PAL Activity Ratio

Example 2 describes the screening of various microorganisms for theirPAL and TAL activities. This information was required to allow forselection of the most suitable microbe for further cloning, expression,purification and kinetic analysis of the PAL and PAL/TAL enzyme.

Medium for growth and induction of PAL in Streptomyces:

A two stage fermentation protocol was used for Streptomyces. Stage Imedium contained, glucose (2%); soybean flour (1%); yeast extract(0.5%); meat extract (0.3%); calcium carbonate (0.3%); used 4% inoculumfor stage II. Stage II medium contained, glucose (2%); yeast extract(2%); sodium chloride (0.5%); calcium carbonate (0.3%). The medium wasdistributed at 100 mL portions into 500 mL flasks. Cells transferred tothis medium were incubated for 24 h at 25° C. on a shaker.

Preparation of cells of Rhodosporidium toruloides following growth inthe Complex Medium:

In order to determine the growth yield and PAL/TAL specific activityRhodosporidium toruloides cells were grown in 50 mL (in 250 mL capacityDeLong flasks) of complex medium. The yield (wet weight of cells) was8.11 grams. The second transfer was made into 200 mL (in 10×one literDeLong flasks) using 0.8 g (wet weight) from the initial harvest. Theyield was 16.0 grams after 3 washes with 100 mM phosphate buffer (pH7.1).

Preparation of Cell Extracts:

The cell pellet was resuspended with 0.5 mL/g cells, 50 mM Tris-HClbuffer (pH 8.5) and disrupted by a single passage through the FrenchPressure Cell at 20,000 psi. The disrupted cells were then centrifugedfor 30 min at 14,200×g to remove the unbroken cell mass. Samples of theextract were used for protein concentration assay and the PAL/TALactivity determination. Protein determination was performed by the BCA(bicinchoninic acid) method from Pierce Co.

Gels:

Precast 7.5% acrylamide gels from BioRad (Cambridge, Mass.) were used.Cell extracts or samples of enzyme solutions were loaded on the gelalong with molecular weight standards were from Pharmacia (Upsula,Sweden). The High Molecular Weight (HMW) lyophilized proteins weresolubilized in 100 μL of 50 mM Tris-HCl pH 8.5 and bromophenol blue wasadded. The running buffer from BioRad was prepared from a 10×solutionand the gels were run at 150 volts. The running dye was electrophoresedoff the gel, the gels were run for an additional 1 h. One end of thegels containing the molecular weight marker and the sample lanes was cutoff and stained with Coomassie blue for approximately 45 min. Twosections of the gel were cut out and the gel material cut up and placedinto 1.0-2.0 mL of 50 mM Tris-HCl buffer pH 8.5 at 4° C. PAL activitywas then measured at two different time intervals. The gel slicescontained a maximum of 173 mU of PAL activity, determined as describedabove.

PAL/TAL Activity:

The PAL/TAL activity was determined as described above. Using thisprocedure, specific activities of 0.0241±0.0005 U/mg and 0.0143±0.0005U/mg were observed for PAL and TAL, respectively (Table 2). Based onthese results, the ratio of PAL/TAL was calculated to be 1.68±0.07. APAL/TAL ratio of 2.12 was observed for the purified enzyme. A literaturevalue of 1.7 has been reported for these enzymes (Hanson and Havir InThe Biochemistry of Plants; Academic: New York, 1981; Vol. 7, pp577-625). The complete data is shown in Table 2.

TABLE 2 PAL and TAL Activity Observed in Cell Free Extracts of VariousMicroorganisms Ratio Specific Activity of ATCC PAL TAL PAL/ # NameMedium (U/mg) (U/mg) TAL 15873 Streptomyces griseus SBG 0.0   ND NDStreptomyces griseus SBG + 0.0004 ND ND Phe Streptomyces griseusStrep. + 0.0003 ND ND Phe 13495 Streptomyces verticillat 1 0.0025 ND ND11386 Sporidiobolus pararoseus 2 0.0158 0.0024 6.51 20804 Rhodotorulagraminis 3 0.0436 0.0102 4.27  2080 Saccharomycopsis fibulige 4 0.00700.0016 4.27 10788 Rhodotorula glutinis 3 0.0241 0.0143 1.68 ND: notdetermined 1. Grown in medium in Can. J Biochem. 48:613-622 (1970) at25° C. 2. Grown in 0.7% Difco malt extract, 0.1% Difco yeast extract and0.1% phenylalanine at 30° C. 3. Grown in 1% malt extract, 0.1% yeastextract and 0.1% phenylalanine medium at 30° C. 4. Grown in 1% peptone,1% yeast extract, 0.5% phenylalanine, 0.1% KH₂PO₄, 0.3% KHPO₄ and 0.05%MgSO₄.7 H₂O at 25° C.

As outlined in Table 2, Rhodosporidium toruloides also known asRhodotorula glutinis (ATCC 10788) possesses the highest TAL activity andwas therefore selected for further studies.

Example 3

Cloning and Expression of Rhodosporidium toruloides PAL in E. coli

Example 3 describes the cloning and expression of phenylalanine ammonialyase (PAL) from Rhodosporidium toruloides in E. coli in order toproduce sufficient quantities of PAL for purification.

RNA Purification:

The Rhodosporidium toruloides RNA was purified from exponential phasecells grown in the complex medium containing phenylalanine. The totalRNA was isolated and the mRNA was purified using Qiagen total RNA andmRNA isolation kits, respectively, according to the manufacturersinstructions.

Reverse Transcription:

The Rhodosporidium toruloides mRNA (3 μL, 75 ng) was reversedtranscribed according to Perkin Elmer (Norwich Conn.) GeneAmp kitinstructions without diethylpyrocarbonate (DEPC) treated water. The PCRprimers used (0.75 μM) were the random hexamers supplied with the kit,the upstream primer (SEQ ID NO:1)5′-ATAGTAGAATTCATGGCACCCTCGCTCGACTCGA-3′ containing a EcoRI restrictionsite, and a downstream PCR primer (SEQ ID NO:2)5′-GAGAGACTGCAGAGAGGCAGCCAAGAACG-3′ containing a PstI restriction site.These were synthesized from the Rhodosporidium toruloides PAL gene. Apositive control using the kit pAW109 RNA and the DM151 and DM152primers was also performed. PCR was carried out for 30 cycles with a 95°C. melting temperature for 1.0 min, a 55° C. annealing temperature for1.0 min and a 72° C. elongation temperature for 2.0 min. Five sec wereadded per cycle to the elongation step and a final elongation step of 10min was used. An aliquot (5.0 μL) was taken from the PCR reaction mixand loaded onto a 1% agarose gel to verify the PCR reaction product.

Digestion of PCR fragments was achieved by using 10×multibuffer (2.0μL), bovine serum albumin (BSA, 10 mg/mL, 1.0 μL), EcoRI and PstI (0.5μL each), PCR product (4.0 μL) and distilled deionized water (12.5 μL).The entire reaction was loaded onto a 1% agarose gel and the desiredsize of the DNA fragments were purified.

Ligation:

The ligation mixture (total vol. 50 μL) for constructs contained:ligation buffer (10×, 5.0 μL), 3.0 U/μL T4 DNA ligase (1.0 μL), BSA (10mg/mL, 2.5 μL), 19 ng/μL PCR product using primers with EcoRI and PstIrestriction sites (25 μL), 33 ng/μL pKK223-3 previously cut with EcoRIand PstI (2.0 μL) and distilled deionized water (14.5 μL). The ligationmixture (total vol. 50 μL) for the control vector contained, ligationbuffer (10×, 5.0 μL), 3.0 U/μL T4 DNA ligase (1.0 μL), BSA (10 mg/mL,2.5 μL), 33 ng/μL pKK223-3 previously cut with EcoRI and PstI (2.0 μL),and distilled deionized water (39.5 μL). The reaction mixtures wereincubated overnight at 16° C.

Transformation:

Competent DH10b E. coli cells (Gibco) were thawed on ice forapproximately 20 min. Then, 2.0 μL of the ligation mix were added to 50μL of the cells and incubated on ice for 30 min. The cells were heatshocked for 20 sec at 37° C. and then chilled on ice again. Then, 0.95mL of LB broth was added to the cells and incubated for 1.0 h at 37° C.on a shaker. The cells were then centrifuged, resuspended inapproximately 50 μL of the LB broth and streaked on LB plates containing100 mg/L ampicillin and incubated overnight at 37° C.

Clones:

The Rhodosporidium toruloides PAL gene was over-expressed in E. coli.The PCR product, which was prepared in this example, was first clonedinto a standard cloning vector and then cloned into pKK223-3over-expression vector under the tac promoter in DH10b E. coli. A totalof six clones were tested for both whole cell and cell free PALactivity.

Cell Growth:

Cells were initially grown overnight, at 37° C. on 50 mL LB media with100 mg/L ampicillin in baffled 250 mL flask. Before harvesting thenon-induced cells, a 5.0 mL aliquot was transferred into the freshmedium and grown to about 0.9 (OD₆₀₀). IPTG was then added to a finalconcentration of 0.2 mg/mL to induce the enzyme and the cells werefurther grown for 3.0 h. OD₆₀₀ measurements are shown in Table 3.

TABLE 3 Growth and PHCA Production mAU's Time Cinn. PHCA [Cinn.] [PHCA](μM/OD₆₀₀) Medium Glucose (hr) OD₆₀₀ 270 nm 10 nm (μM) (μM) Cinn. PHCALB 0    24 ND 10689.27 5000.41 839.42 629.50 LB 0.2% 24 ND 12000.603540.89 942.40 445.76 M9 0.2% 24 0.2377 418.84 336.98 32.89 42.42 138.37178.47 M9 2%   24 0.2079 411.03 370.11 32.28 46.59 155.26 224.11 LB 0   72 2.1010 12698.07 7942.28 997.17 999.85 474.62 475.89 LB 0.2% 72 4.274014038.05 8416.65 1102.40 1059.57 257.93 247.91 Cinn. 12734.13 PHCA7943.46

Whole Cell PAL Activity:

Aliquots of non-induced (1.0 mL) and induced (0.2 mL) cells were takenbefore harvest. The cells were pelleted and resuspended in 1.0 mL of 50mM Tris buffer pH 8.5. Phenylalanine was then added (1.0 mM, finalconcentration) and the mixtures were incubated on the shaker at 37° C.for 1.0 h. The mixtures were then acidified with 50 μL of phosphoricacid and the cells were pelleted. The solute was then filtered andanalyzed by HPLC as described above. The culture media from the inducedcells was similarly treated and analyzed by HPLC. Cinnamic acid and PHCAstandards (1.0 mM) were also analyzed and used to calculate theconcentration of the compounds in the samples (e.g., (177.66 mAUsample)/(12734.13 mAU/mM standard)*(1000 μM/mM)=13.95 μM cinnamic acid).Results are shown in Table 3.

Cell Free PAL Activity:

Cells were harvested by centrifugation. To the harvested cell pellet,1.0 mL of 50 mM Tris buffer (pH 8.5) was added and the cells weredisrupted by a single passage through the French Pressure Cell atapproximately 18,000 psi. The extract was centrifuged for 10-15 min inan Ependorf Microfuge at 4° C. The supernatant (1.0 mL) was removed andused for PAL activity and Bradford protein assays (Bradford, M., Anal.Biochem., 72, 248, 1976). The highest specific activities observed, were0.244 (PAL) and 0.0650 (TAL) U/mg protein.

SDS Gel Electrophoresis:

The purified PAL protein was run on a 8-25% native PhastGel as describedin General Methods. The molecular weight of the purified PAL wasestimated to be 287 kD based on these analyses.

During the above experiments, it was discovered that both PHCA andcinnamic acid appeared during growth of the cells in the LB medium andalso during the whole cell assays. Detection of PHCA in transformed E.coli cultures was an unexpected discovery since E. coli does not containthe enzymatic machinery for conversion of cinnamic acid to PHCA.Presence of PHCA in these cultures therefore indicated that the wildtype yeast PAL enzyme expressed in E. coli, in addition to its PALactivity, contained the TAL activity and directly converts tyrosine toPHCA.

Example 4

PHCA Production from Glucose by Recombinant E. coli Over-Expressing theRhodosporidium toruloides Wild Type PAL

This Example describes analysis of the E. coli strain over-expressingthe wild type PAL for its ability to produce PHCA during growth ineither glucose or the LB medium.

As described above, there are two pathways to synthesize PHCA. In onepathway, PHCA can be synthesized through conversion of phenylalanine byPAL to trans-cinnamic acid which is in turn hydroxylated at the paraposition by the cytochrome P-450 enzyme system. In the other pathway,tyrosine is converted to PHCA in a single step reaction by TAL and nocytochrome P-450 is required. Since no cytochrome P-450 enzyme ispresent in E. coli, any PHCA formed in these cells should be through theTAL route. To confirm this hypothesis, the following experiments werecarried out: E. coli cells containing PCA12Km (described in Example 8)were incubated overnight with 1.0 mM cinnamic acid, and the PHCAproduction was monitored by HPLC.

Cell Growth:

The cells were grown overnight, in LB broth, LB+0.2% glucose with 100mg/L ampicillin at 30° C. or in the M9 medium (see below)+0.2% glucoseor the M9 medium+2% glucose with 100 mg/L ampicillin for 24 h at 30° C.The cells grown in the M9 medium+glucose grew significantly more slowlythan the cells in the LB medium.

Assay of PHCA:

An aliquot (1.0 mL) of each cell culture was acidified with 50 μL ofphosphoric acid and pelleted and the supernatant was filtered andanalyzed by HPLC as described in the General Methods. Samples were takenafter 24 or 72 h. A PHCA standard (1.0 mM) was also analyzed and used todetermine the concentration of the compound in the samples. Samples werealso taken to measure the cell density at 600 nm in order to relategrowth to PHCA production (see Table 3, Example 2).

As can be seen from the data in Table 3, the E. coli cells containingthe wild type PAL produced PHCA when grown in either the LB (with andwithout glucose) or M9 (see below) with glucose medium. The addition ofglucose to the LB medium increased the total amount of PHCA formed andthe cell density of the culture, but decreased the PHCA production percell density.

M9 medium:

The M9 minimal medium for culturing bacteria contains (in gram perliter): Na₂HPO₄, 6.0; KH₂PO₄, 3.0; NH₄Cl, 1.0; NaCl, 0.5; and glucose,4. (Maniatis, Appendix A.3).

Example 5

Purification of the Recombinant Wild Type Rhodosporidium toruloides PALfrom E. coli

The wild type recombinant R. toruloides PAL from transformed E. coli waspurified using heat treatment, ammonium sulfate precipitation, anionexchange column, and hydrophobic interaction chromatography and gelfiltration.

Cell Growth:

The cells were grown in a 10-L fermenter at 28° C. on 2×YT medium with100 mg/L ampicillin.

Preparation of Cell Free Extracts:

The cells were harvested and kept as a frozen pellet until required foruse. The pellet (76 g wet weight) was washed with 50 mM Tris-HCl pH 8.5and resuspended with the same buffer to a density of 2.0 g wet weight ofcells per 1.0 mL of buffer. A small amount of DNase was added and thecells were passed twice through a French Pressure Cell at approximately18,000 psi. The protease inhibitor, PMSF, was then added to the extractto a final concentration of 0.5 mM. The cell debris was removed bycentrifugation at 13,000×g for 30 min followed by another centrifugationat 105,000×g for 1.0 h.

Heat Treatment of Extract:

The extracts were heated to a temperature of 60° C. for 10 min and thenplaced on ice. The denatured proteins were pelleted by centrifugation at25,000×g for 30 min.

Ammonium Sulfate Precipitation:

Ammonium sulfate precipitation was achieved by addition of saturatedsolutions of ammonium sulfate at 4° C. The solution was stirred on icefor 15-30 min. The precipitated protein was pelleted by centrifugationat 25,000×g for 15 min and the pellet dissolved in a minimal amount ofTris buffer. During the 35% ammonium sulfate cut, the pH of the solutionwas measured and adjusted back to 8.5. The volume of the extract wasmeasured after each precipitation. The extracts were ammonium sulfateprecipitated separately, but the 50% ammonium sulfate cuts from bothruns were pooled, concentrated and desalted with Centricon-50ultrafiltration tubes (Milipore, Bedford, Mass.).

Anion Exchange Chromatography:

Anion exchange chromatography was carried out on a 20 mm×165 mm, 50 μmHQ column (Perseptive Biosystems, Farmingham, Mass.) at a flow of 30mL/min. The starting buffer (buffer A) was 5 mM Tris-HCl pH 8.5 and theeluting buffer (buffer B) was 0.5 M NaCl in 5.0 mM Tris-HCl pH 8.5. Thecolumn was equilibrated for two column volumes (CV) and washed for twoCV with buffer A after sample injection. A gradient was run from 100% ofbuffer A to 50% of buffer A and buffer B over 10 CV. A second gradientwas then run from 50% of buffer A and buffer B to 100% of buffer B overtwo CV. The column was washed with two CV of buffer B and thenre-equilibrated with buffer A for two CV. Protein was monitored at 280nm and 10 ml fractions were collected on ice during the first gradient.The sample size was up to 5.0 ml and contained up to 340 mg of proteinor approximately 12% of the column's capacity of 2850 mg. Fractions fromthe different runs were pooled and concentrated as indicated above.

Hydrophobic Interaction Chromatography:

Hydrophobic interaction chromatography was carried out on a 20 mm×167mm, 50 μm PE column (Perseptive Biosystems, Farmingham, Mass.) at a flowrate of 30 mL/min. The starting buffer (buffer A) was 1.0 M (NH₄)₂SO₄ in5.0 mM Tris-HCl pH 8.5 and the eluting buffer (buffer B) was 5.0 mMTris-HCl pH 8.5. The column was equilibrated for two CV and washed fortwo CV with buffer A after sample injection. A gradient was then runfrom 100% of buffer A to 100% of buffer B over 10 CV. The column wascleaned with 2 CV of buffer B and then re-equilibrated with buffer A fortwo CV. Protein was monitored at 280 nm and 10 mL fractions werecollected and kept on ice during the gradient. Samples, up to 5.0 mL andcontaining up to 50 mg of protein or 12% of the column's capacity of 420mg, were adjusted to 1.0 M (NH₄)₂SO₄ by the addition of a saturatedammonium sulfate solution. Fractions from different runs were pooled,desalted and concentrated as indicated above.

Gel Filtration Chromatography (GF):

Gel filtration was carried out on a 10 mm×305 mm, Superdex 200 HR columnat a flow rate of 0.5 mL/min. Using a 50 mM Tris-HCl buffer (pH 8.5)containing 0.2 M NaCl, a column was run for one CV and protein elutionmonitored at 280 nm. Fractions (0.5 mL) were collected and kept on ice.The volume of the sample applied to the column was 100 μL and containedup to 10 mg of protein. The fractions from the center of the peaks werepooled and concentrated as described above.

Data describing the purification and increase in specific activity areshown in Table 4.

TABLE 4 Purification of PAL from E. coli Total PAL Total Specific Vol.Protein Protein Activity Activity Activity Yield Purif. Step (mL)(mg/mL) (mg) (U/mL) (Units) (U/mg) (%) (Fold) Crude 70 77.4 5415 13.62953.6 0.176 100%  1.00 Extract Heat 43 52.0 2237 14.95 642.8 0.287 67%1.63 Treatment Am. Sulf. 22.2 55.6 1235 54.7 1213.6 0.982 127% 5.58 PPTAnion 15.0 12.7 190.2 55.63 834.4 4.387 88% 24.92 Exchange HIC 5.8 15.188 113.75 659.7 7.530 69% 42.76 Gel 4.4 8.9 39 54.77 241.0 6.143 25%34.89 Filtration GF Ends 1.6 6.6 11 31.47 50.3 4.774  5% 27.11

Example 6

Carbon Source Selection

This example describes the effect of various carbon sources on theability of the recombinant Saccharomyces cerevisiae strain (PTA 408)containing the Rhodosporidium toruloides PAL gene (SEQ ID NO:7) plus theplant P-450 and the P-450 reductase (SEQ ID NO:11 and SEQ ID NO:13respectively) to convert phenylalanine to PHCA.

Two colonies (#1 and #2) from the Saccharomyces cerevisiae containingthe yeast PAL and the plant cytochrome P-450 and the cytochrome P-450reductase were chosen and grown on three different media containingeither raffinose, galactose or glucose. The media were identified asRaf/SCM or Gal/SCM or Glu/SCM. The formulation of various media used inthese experiments is indicated below:

Glu/SCM (Ade/His/Ura) contained: Bacto-yeast nitrogen base (6.7 g/L);glucose, (20.0 g/L); and SCM, (2.0 g/L).

Raf/SCM(Ade/His/Ura) contained: Bacto-yeast nitrogen base, (6.7 g/L);raffinose, (20.0 g/L); and SCM, (2.0 g/L).

Raf/Gal SCM (Ade/His/Ura)/Tyr/Phe contained: Bacto-yeast nitrogen base,(6.7 g/L); raffinose, (10.0 g/L); galactose, (10.0 g/L); SCM, (2.0 g/L);tyrosine, (0.5 g/L); and phenyalanine, (10.0 g/L).

SCM (Ade/His/Ura) agar plate for yeast contained: Bacto-yeast nitrogenbase, (3.35 g/L); dextrose, (10.0 g/L); agar, (10.0 g/L); SCM, (1.0g/L); and ddH₂O, (500 mL).

Glycerol stocks (300 μL) of each of the colonies were used to inoculatethe Glu/SCM, Gal/SCM and Raf/SCM media. Duplicate cultures were preparedwith each strain and each medium and cultures were grown for 24 and 48h.

The cell density was measured as described above and the cells were thencentrifuged, washed once with 0.85% saline phosphate buffer, resuspendedin the SCM medium (5.0 mL) and the OD₆₀₀ was measured again. The cellswere then added to the corresponding flasks which contained either 25.0mL of the Raf/SCM or the Gal/SCM medium to the final OD₆₀₀ of 0.5.Galactose (5% final concentration) was added to each flask and left onthe shaker for about 16 h to allow for induction. Following induction,phenylalanine (1.0 mM final concentration) was added to each flask andsamples (1.0 mL) were taken from each flask at 2, 4, 6, 24 and 48 h andanalyzed by HPLC for the presence of PHCA (see Table 5).

TABLE 5 Carbon Source 2h 4h 6h 24h 48h Strain #1 PHCA Production (μM)after Addition of 1.0 mM Phenylalanine raffinose 116.28 125.43 165.34270.15 99.85 galactose 95.54 164.71 183.51 231.25 97.56 glucose 57.42128.04 170.18 269.62 91.71 Strain #2 PHCA Production (μM) after Additionof 1.0 mM Phenylalanine raffinose 145.95 188.06 218.45 293.54 116.7galactose 150.85 171.07 196.26 230.95 103.09 glucose 75.71 179.52 161.1238.77 78.65

As is seen by the data in Table 5, both strains tested appeared tobehave similarly when grown in different media. The highest level ofproduction of PHCA was observed between 6-24 h and around 30% of thephenylalanine was converted to PHCA. Following the initial appearanceand accumulation, a decrease in the concentration of the PHCA wasobserved (48 h).

Example 7

Production of PHCA by Recombinant Saccharomyces cerevisiae StrainContaining the Rhodosporidium toruloides PAL, the Plant

Cytochrome P-450 and the Cytochrome P-450 Reductase

This example describes induction by galactose for production of PHCA bya recombinant Saccharomyces cerevisiae strain that contains the wildtype PAL plus the plant cytochrome P-450 and the cytochrome P-450reductase genes.

Since PAL, the cytochrome P-450 and the cytochrome P-450 reductase thathad been incorporated into the Saccharomyces cerevisiae strain, wereunder the control of the galactose promoter, experiments were performedin order to examine the effect of the length of induction by galactoseon the level of PHCA formed. Saccharomyces cerevisiae strain #2, whichhad produced the highest level of PHCA, was chosen and induced bygalactose. In order to examine if the recombinant Saccharomycescerevisiae could directly convert glucose to PHCA, one set of cells,after one h induction with galactose, received glucose but nophenylalanine was added. Another set of cells was grown on raffinose.Samples were taken from all flasks at intervals and prepared for HPLCanalysis as described above.

A sample of the glycerol suspension of Saccharomyces cerevisiae wasstreaked on an SCM-glucose plate and incubated at 30° C. Four colonieswere picked from the plate, inoculated into 4.0 mL of Glu/SCM mediumleft on the shaker (30° C., 250 rpm) overnight. One mL of the cellsuspension was taken and the OD₆₀₀ measured. After 24 h of growth, whenthe OD₆₀₀ was around 1.4 to 1.6, cells (1.0 mL) were transferred to 25mL of Glu/SCM medium or 50 mL of Raf/SCM medium. Following overnightgrowth (30° C., 250 rpm) samples 1.0 mL) were taken from each flask andthe OD₆₀₀ measured.

OD₆₀₀ 1. #1 in Raf/SCM medium 0.3775 2. #1 in Glu/SCM medium 1.5119 3.#2 in Raf/SCM medium 0.4730 4. #2 in Glu/SCM medium 1.4923

As can be seen from the OD data, higher cell mass was obtained aftergrowth on glucose compared to raffinose. In order to examine if therecombinant Saccharomyces cerevisiae could directly convert glucose toPHCA without additional phenylalanine an experiment was set up in whichfollowing growth on glucose or raffinose, cells were induced bygalactose prior to glucose addition.

Samples were taken at intervals and prepared for HPLC analysis asdescribed above. Data is shown in Table 6.

TABLE 6 Effect of Growth on Glucose Versus Raffinose on PHCA Productionby Saccharomyces cerevisiae Containing PAL + P-450 + P-450 Reductase(induced by galactose) PHCA production (μM) Induction Incubation Time1.0 h Induction 3.0 h Induction 6.0 h Recombinant Saccharomycescerevisiae grown on glucose:  2.0 h 0.33 1.37 4.58  4.0 h 0.57 2.62 N/A 6.0 h 0.48 3.33 N/A 24 h 0.98 7.63 14.59 48 h 2.14 6.64 14.25Recombinant Saccharomyces cerevisiae grown on raffinose  2.0 h 7.1130.72 62.73  4.0 h 9.33 45.49 N/A  6.0 h 12.55 55.88 N/A 24 h 28.49112.37 202.77 48 h 38.63 110.85 193.63

As shown in the data in Table 6, while growth in the medium containingglucose produced higher cell mass, the amount of PHCA formed was muchhigher following growth in the presence of raffinose (approximately 200μM PHCA within 24 h following growth in raffinose versus approximately14.5 μM following growth in glucose). This underlines the inhibitoryeffect of glucose on the galactose inducible promoter.

In another experiment, the effect of addition of phenylalanine to thegrowth medium containing either glucose or raffinose was determined.Samples (10 mL) from each flask were transferred into a 125 mL capacityflask containing 25 mL of medium and cells were induced by galactose (2%final concentration). The induction was allowed for 1.0, 3.0, 6.0 h andovernight. After the specified induction time, phenylalanine (1.0 mM)was added to each flask and formation of PHCA was measured. Results aresummarized in Table 7 below.

TABLE 7 Effect of Addition of Phenylalanine on PHCA Production bySaccharomyces cerevisiae Containing PAL + P-450 + P-450 Reductase DuringGrowth on Glucose Versus Raffinose (induced by galactose) PHCAProduction (μM) after Addition of 1.0 mM Phenylalanine Induction CarbonTime Source 2.0h 4.0h 6.0h 24h 48h 54h 1.0h raffinose 9.33 45.05 57.69475.62 531.25 554.46 glucose 1.65  7.01  5.99 172.04 233.97 230.24 3.0hraffinose 37.66 72.44 155.16  459.99 536.54 545.29 glucose 4.14  4.8618.55 212.02 318.62 342.95 6.0h raffinose 142.02 N/A N/A 454.71 539.33537.62 glucose 25.21 N/A N/A 235.42 369.9 372.24 overnight raffinose11.56 208.11  260.46  497.97 424.28 408.32 glucose 9.87 10.13 36.74354.15 424.89 398.81

As depicted in Table 7 and as expected, addition of phenylalanine toboth cultures resulted in production of higher levels of PHCA comparedto those produced in the absence of additional phenylalanine. Generallycells grown on raffinose produced higher amounts of PHCA fromphenylalanine compared to those grown on glucose. The average level ofPHCA produced from phenylalanine by cells growing on raffinose wasaround 500 μM and the highest level of PHCA was formed at around 24 h.In most cases, the level of PHCA reached a maximum at or around 24 h andremained without significant changes until the end of the experiment(approximately 54 h). Duration of induction (i.e., 1.0, 3.0, 6.0 h andovernight) did not seem to make a significant difference in the level ofPHCA production. The level of PHCA formed in cultures growing on glucosewas around 300 μM. While the total amount of PHCA formed byglucose-grown cells was less than that produced by cells grown onraffinose, the pattern of production of PHCA was similar.

In summary, the recombinant Saccharomyces cerevisiae cells containingthe Rhodosporidium toruloides wild type PAL plus the plant cytochromeP-450 and the cytochrome P-450 reductase had the ability to convertglucose directly, in the absence of additional phenylalanine, to PHCA(approximately 25% conversion). When phenylalanine was added around 50%was converted to PHCA.

Example 8

Development of a Selection System for Identification of the Mutant PAL(PAL/TAL) Enzyme

This example describes a method for selection of the mutant PAL enzymewith improved TAL activity. There are currently no engineered enzymesthat can efficiently catalyze the conversion of tyrosine directly toPHCA with no intermediate step. In this reaction, the enzyme convertstyrosine to PHCA and ammonia while in the reverse reaction the sameenzyme converts PHCA and ammonia to tyrosine. In order to detect mutantPAL enzymes able to convert tyrosine to PHCA, the following screen wasdeveloped.

Constructing the expression vector (PCA12Km):

A weak expression vector was made by modifying the commerciallyavailable pBR322 vector. Briefly, pBR322 was digested with Pst I andsubjected to 20 cycles of PCR with two primers, pBR1 (SEQ ID NO:3)5′-GAGAGACTCGAGCCCGGGAGATCTCAGACCAAGTTTACTCATATA-3′ and pBR2 (SEQ IDNO:4) 5′-GAGAGACTCGAGCTGCAGTCTAGAACTCTTTTTTCAATATTATTG-3′. The PCRreaction product was extracted with phenol chloroform, EtOH precipitatedand digested with Xho I. The Xho I digested product was then gelisolated, ligated and transformed into E. coli selecting fortetracycline resistance. This vector is therefore a pBR322 lacking theampicillin resistance gene but containing the beta-lactamase promoterand the following restriction sites: Xba I, Pst I, Xho I, Sma I, Bgl II.The tetracycline-resistance gene in pBR322 was replaced by thekanamycin-resistance gene. Tetracycline resistant gene was cut out ofpCA12 (FIG. 1) at EcoR V (185) and Nru 1 (972) sites, the ends werepolished by using pfu polymerase (PCR polishing kit, Stratagene) andligated with the blunt-ended 9 kb kanamycin resistant gene fragment(Vieira and Messing, Gene 19:259-268 (1982)). Final construction wasselected on the LB/km plates after transformation of the ligation in tothe XL1-Blue vector (FIG. 1).

Subcloning the Rhodosporidium toruloides PAL gene into PCA12Km:

The gene sequence of yeast (Rhodosporidium toruloides) PAL has beendetermined and published (Anson et al., Gene 58:189-199 (1987)). Basedon the published sequence, the gene was subcloned into a pBR322-basedvector. The entire PAL gene was then removed from the plasmid byXbaI-PstI digestion, and the gene was ligated into theXbaI-PstI-digested PCA12Km. The new construct containing the PAL genewas designated PCA18Km.

Expression of the PAL Enzyme in the Tyrosine-auxotrophic E. coli:

The PCA12Km and PCA18Km were transformed into the tyrosine-auxotrophicE. coli strain AT2471 and the TAL and PAL activities were measured usingthe whole cell assay. Formation of small quantities of PHCA or cinnamicacid were detected following incubation of these cells with tyrosine orphenylalanine (Table 8).

TABLE 8 PHCA and Cinnamic Acid Formation from Tyrosine andPhenylalanine, Respectively* PHCA (μM) cinnamic acid (μM) Cellscontaining PCA12Km 0 0 Cells containing PCA18Km 10.8 38 *The cells wereincubated with 1.0 mM tyrosine or phenylalanine for 1.0 h, and the PHCAor cinnamic acid formation was detected by HPLC (see general methods).

As seen in Table 8, the yeast PAL enzyme was weakly expressed in thetyrosine-auxotrophic E. coli strain AT2471 containing the PCA18Kmvector.

Determination of selection condition (Development of a selectionsystem):

The tyrosine-auxotrophic E. coli strain AT2471 containing pCA18Km showedthe same tyrosine-auxotrophic property as the original strain. The cellsdid not grow on the minimal plate, but grew well when 0.004 mM tyrosinewas added to the plate. In order to find the suitable condition forselection, cell growth was tested on a minimal plate containing variousconcentrations of tyrosine or PHCA (see Table 9).

TABLE 9 Tyrosine-Auxotrophic E. coli Cell Growth Under VariousConcentrations of Tyrosine or PHCA Tyrosine* PHCA** 0.0001-0.0002 mM −+++ 0.0003-0.0005 mM + +++ 0.001-0.004 mM +++ +++ 1.0-2.0 mM ++ 4.0-6.0mM + 10 mM − *No PHCA was added in the growth medium for testing thetyrosine-auxotrophic property. **0.004 mM tyrosine was added in thegrowth medium for testing the toxicity of PHCA. −: no growth; +: verypoor growth; ++: poor growth; +++: good growth.

The results shown in Table 9 indicate that high concentrations of PHCAare toxic to the cells. The 2.0 mM PHCA concentration was chosen for theselection experiment. The information about the cell growth at differenttyrosine concentrations is important for the selection. For example,when the tyrosine made from PHCA by the cell is not enough to supportcell growth, small amounts of tyrosine (0.0001-0.0002 mM) can be addedto the plate. This will allow identification of strains expressing theenzyme with slightly improved TAL activity. In other words, theselection stringency can be modulated by changing the concentration ofPHCA and tyrosine in the selection plate.

Example 9

Engineering the Mutant PAL Enzyme with Improved TAL Activity

Error-Prone PCR:

The following primers were used for amplifying the entire PAL gene fromPCA18Km construct:

Primer A (SEQ ID NO:5):

5′-TAGCTCTAGAATGGCACCCTCG-3′

Primer B (SEQ ID NO:6):

5′-AACTGCAGCTAAGCGAGCATC-3′.

The primer A (forward primer) contained a Xba I restriction enzyme sitejust before the ATG codon, and primer B (reverse primer) had a Pst Isite just after the stop codon. To increase the rate of the PCR error,plain Taq polymerase and more reaction cycles (35 cycles) were used. Inaddition, the ratio of dATP, dTTP, dGTP and dCTP was changed. Fourdifferent reactions were performed. In each reaction, the concentrationof one of the dNTP's was 0.1 mM, and the other three dNTP were adjustedto 0.4 mM. The PCR products from four reactions were mixed togetherfollowing completion of the reaction. After digestion of the error-pronePCR products with Xba I and Pst I, fragments were ligated into theXbaI-PstI-digested PCA12Km.

In vivo mutagenesis using E. coli XL1-Red strain:

The PCA18Km was transformed into the XL1-Red strain, and the cells weregrown overnight in the LB medium plus kanamycin. To increase themutation rate, the cells were diluted with fresh growth medium, andgrown further. After 2-4 cell generation cycles, the plasmids werepurified, and used for selection.

Selection:

After mutagenesis, the pool of mutated PCA18Km containing the randomlymutated PAL gene were transformed into the tyrosine-auxotrophic E. colistrain by electroporation. The transformation efficiency was 1.5×10⁸cfu/μg DNA. The cells were then incubated in the LB medium withantibiotics for more than 5.0 h. After washing with the minimal medium,the cells were streaked on plates containing the minimal mediumsupplemented with 0.0002 mM tyrosine and 2.0 mM PHCA. After 3.0-5.0 daysof incubation at 30° C., 1.0-10 colonies appeared on each plate.

The colonies that had appeared on the selection plates were analyzed fortheir PAL/TAL activity using the whole cell assay described in thegeneral methods. One of the mutants, designated EP18Km-6, showed anenhanced TAL activity ratio than the wild type cell. Genetic analysisfor the EP18Km-6 mutant was carried out. The plasmid DNA was purifiedfrom the mutant cells, and then re-transformed into E. coli. The newtransformant showed the same enhanced TAL ratio as the original mutant,indicating that all mutations that involved improvement of TAL activitywere on the plasmid. To better characterize the mutants, the followinganalyses were carried out.

Example 10

Characterization of the Mutant PAL Enzyme

Sequence analysis of the mutant gene:

The entire gene of EP18Km-6 was sequenced on an ABI 377 automatedsequencer (Applied Biosystems, Foster City, Calif.), and the datamanaged using DNAstar program (DNASTAR Inc., Madison, Wis.). Analysis ofthe resulting PAL mutants followed by comparison with the wild type gene(SEQ ID NO:7), indicated that the mutant gene (SEQ ID NO:9) containedthe following four single base substitution mutations (point mutations):CTG (Leu215) to CTC, GAA (Glu264) to GAG, GCT (Ala286) to GCA and ATC(Ile540) to ACC. The first three mutations were at the third base,generating silent mutations which did not result in any amino acidchange. The fourth mutation was a second base change (ATC to ACC). Thismutation changed the isoleucine-540, which is in the conserved region ofthe enzyme, to a threonine. Various PAL enzymes from different sourceshave either isoleucine or leucine at this critical position.

Over-expression and purification of EP18Km-6 mutant enzyme:

In order to obtain sufficient quantities of the pure enzyme forenzymatic kinetics analysis, the enzyme was expressed in theover-expression vector, pET-24-d. The pET-24-d vector was digested withEcoRI, and the digestion product was filled-in using the Klenow enzyme(Promega, Madison, Wis.) according to the manufacturer's instructions.The linearized vector was then digested with NheI and the mutant PALgene was obtained by cutting the EP18Km-6 with XbaI and Smal. Since theNheI and XbaI are compatible sites, the mutant gene was subcloned intothe pET24-d vector by ligation in order to prepare the pETAL construct(FIG. 2). Although the pET-24-d vector carries an N-terminal T7 Tagsequence plus an optional C-terminal HisTag sequence, these Tags werenot used so that the enzymes could be expressed with natural sequencesat both N- and C-termini. After cloning the mutant gene into thepET-24-d vector, the construct was transformed into E. coli BL21(DE3).For over-expression, the cells were grown in the LB medium containingkanamycin to an OD₆₀₀ of 1.0 before 1.0 mM IPTG was added. After 4.0 hof induction, cells were harvested by centrifugation and the crudeextracts prepared as described in the General Methods section. TheSDS-PAGE analysis of the crude extracts revealed that the expressedenzyme was the dominant protein band, and the expression level wasestimated to be 10-15% of total protein (FIG. 3). FIG. 3 shows theSDS-PAGE of purified mutant PAL enzyme (lane A) and the cell crudeextracts (lane B) which has been used as the starting materials forpurification. Lane C is the standard marker of molecular weight (94, 67,43, 30, 20 and 14 kDa from top to bottom). For purification of PAL, thecell pellet was suspended in 10 mM potassium phosphate buffer, pH 6.6,containing protease inhibitors PMSF, amino caproic acid and benzamidine(1.0 mM, each). The cells were broken by sonication (Branson model 185,70% power setting, 4 min in ice bath), followed by centrifugation(30,000×g, 30 min). The clear supernatant was applied to a Mono-Q HPLCcolumn (flow rate of 1.0 mL/min). The column was started using a 50 mMpotassium phosphate buffer, pH 7.0, followed by a 400 mM potassiumphosphate buffer, pH 7.2 as the elution buffer. The enzyme was eluted ata concentration of approximately 90 mM potassium phosphate. The activefractions were pooled and concentrated using Centricon YM100 (Milipore,Bedford, Mass). The enzyme was >98% pure as judged by the SDS-PAGEelectrophoresis (FIG. 3).

Biochemical characterization of mutant PAL enzyme:

A detailed enzyme kinetics analysis using the yeast wild type and thepurified mutant PAL enzyme and tyrosine or phenylalanine as substrate,was carried out. The PAL and TAL activities were measuredspectrophotometrically as described in the General Methods and the K_(M)and V_(max) were determined from its Lineweaver-Burke plot. The k_(cat)was calculated from V_(max) assuming the presence of four active sitesin the active tetramer. The determined K_(M) and k_(cat) of the enzymesare shown in Table 10.

TABLE 10 K_(M) and k_(cat) of wild type and mutant PAL Enzyme K_(M) (mM)k_(cat) (sec⁻¹) WT (Phe)* 0.250 2.09 WT (Tyr)** 0.111 0.46 Mutant (Phe)*0.333 2.45 Mutant (Tyr)** 0.05 0.63 *Phenylalanine was used assubstrate. **Tyrosine was used as substrate.

The catalytic efficiency, defined as k_(cat)/K_(M), was calculated forboth the wild type and mutant enzyme. The ratio of the TAL catalyticefficiency versus the PAL of the wild type enzyme was 0.5 while that ofthe mutant enzymes had increased to 1.7 (see Table 11). The resultsshowed that unlike the wild type PAL enzyme which preferred to usephenylalanine, the mutant enzyme preferred to use tyrosine as substratethereby clearly demonstrating that the substrate specificity of theyeast PAL enzyme had changed after mutagenesis and selection.

TABLE 11 Catalytic Efficiency¹ and TAL/PAL Ratio Comparison Between WildType and Mutant PAL PAL(k_(cat)/K_(M)), TAL(k_(cat)/K_(M)), EnzymeM⁻¹sec⁻¹* M⁻¹sec⁻¹** TAL/PAL ratio Wild type 8.36 × 10³  4.14 × 10³ 0.5Mutant 7.36 × 10³ 12.61 × 10³ 1.7 *Phenylalanine was used as substrate**Tyrosine was used as substrate ¹catalytic efficiency is defined ask_(cat)/K_(M)

Example 11

Bioproduction of PHCA from Glucose in E. coli through the TAL Route

Production of PHCA from glucose in E. coli using the mutant PAL:

The plasmid with the mutant PAL gene, with improved TAL activity (EP18Km-6), was transformed into the wild type E. coli W3110. The cellswere then grown in the minimal medium using glucose as the sole carbonsource. After overnight growth, 20 μL of the growth medium was filtered,and analyzed by HPLC for detection of PHCA. No PHCA accumulation wasfound in the wild type (control) E. coli cells. However, when the mutantPAL gene was expressed in E. coli, 0.138 mM PHCA was detected in theovernight growth of the cells in a minimal medium containing glucose asthe sole carbon source (see Table 12).

TABLE 12 PHCA Production from Glucose in E. coli cells With and WithoutMutant PAL/TAL Enzyme Expressed PHCA (μM) Cinnamic acid (μM) Wild typeE. coli 0 0 E. coli with mutant PAL expressed 135 90

The E. coli Cells Lack Cinnamate Hydroxylase Activity:

An overnight incubation of the E. coli cells, containing the PCA12Kmvector, with 1.0 mM cinnamic acid did not result in PHCA productionunderscoring the lack of ability of the E. coli cells to convertcinnamic acid to PHCA.

Example 12

Incorporation of the Modified PAL into the Yeast Expression Vector

The following yeast strains and the pGPD316 expression vector were used.Strain ZXY34-1A contained the genotype: Mata, ade2-1, can1-100, his3-11,-15, leu2-3, -112, trup1-1, ura3-1, aro 3:: ΔURA3, aro4:: ΔHIS3 and wasdesignated an aro3, aro4 double knockout. Strain ZXY0304A contained thegenotype: Mata, ade2-1, can1-100, his3-11, -15, leu2-3, -112, trup1-1,ura3-1, aro 3:: Δura3, aro4:: ΔHIS3 was an aro3, aro4 double knockout.

Using standard sub-cloning methods well known in the art, the modifiedPAL was incorporated into the above vectors. The modified PAL cDNA (2.0kb) was cut by XbaI and SmaI restriction enzymes and the cut fragmentobtained ligated into the expression vector pGPD316 which had been cutby SpeI and SmaI restriction enzymes. The new construct from pGPD316plus insert from pEp18 was designated pGSW18 (FIG. 4). The new constructwere verified by restriction enzyme digestion followed by agarose gelelectrophoresis.

Example 13

The Ability of ARO4GSW to Convert Glucose to PHCA in the Absence ofAromatic Amino Acids

Strain ZXY0304A contained the genotype: Mata, ade2-1, can1-100, his3-11,-15, leu2-3, -112, trup1-1, ura3-1, aro 3::Δura3, aro4::ΔHIS3,designated as an aro3, aro4 double knockout was used. The ZXY0304Astrain was transformed by pGSW18 containing the modified PAL with thestandard lithium acetate method. The transformants, ARO4GSW, wereselected using the SCM medium (without leucine and uracil). ARO4GSW (100μL glycerol stock) was used to inoculate the regular SCM mediumcontaining 2% glucose. The organisms were grown at 30° C. for 5.0 h,cells were centrifuged, resuspended in the SCM medium containing 2%glucose but without aromatic amino acids and allowed to grow overnight.The cells were then centrifuged and resuspended in the following mediato a final cell density of 1.0 (OD₆₀₀ nm): a) regular SCM mediumcontaining about 400 μm of phenylalanine and tyrosine and b) SCM mediumwithout aromatic amino acids. The cells were left on the shaker (250rpm, 0° C.) and samples (1.0 mL) were taken for HPLC analysis at 2.0,4.0, 6.0 and 16 h. The results are shown in Table 13.

TABLE 13 Effect of Aromatic Amino Acids on PHCA Production of AR04GSWYeast Strain PHCA production (μM) 2.0 h 4.0 h 6.0 h 16 h no aromatic AA*1.157 2.187 2.866 5.813 aromatic AA 3.806 6.316 10.313 15.147 *AA =amino acid

As shown in Table 13, the recombinant yeast strain ARO4GSW produced PHCAfrom glucose in the absence of any additional aromatic amino acids inthe growth medium. The data also demonstrate that addition of aromaticamino acids to the growth medium results in almost a 2.5 fold increasein the level of PHCA produced compared to growth in the absence ofaromatic amino acids. These results underscore the ability of therecombinant Saccharomyces cerevisiae containing the mutated PAL, in theabsence of the cytochrome P-450 and the cytochrome P-450 reductase, toconvert glucose to PHCA.

Example 14

Effect of Phenylalanine and Tyrosine on PHCA Production by ARO4GSW

Containing the Modified PAL During Aromatic Amino Acid Starvation

This Example investigates the effects of phenylalanine and tyrosine onPHCA production by the recombinant yeast strain ARO4GSW containing themodified PAL during aromatic amino acid starvation.

A sample (100 μL) of glycerol stock of ARO4GSW was inoculated into theregular SCM medium containing 2% glucose. The cells were left on theshaker at 30° C. for 5.0 h before they were harvested. The pellet wasresuspended in the SCM medium containing 2% glucose but without aromaticamino acids and grown overnight at 30° C. on a shaker. The cultures werethen centrifuged and resuspended in the following media with final celldensity of 1.0 OD₆₀₀ an: a) regular SCM medium, b) SCM medium containingno aromatic amino acids, 2% glucose and 1.0 mM phenylalanine and c) SCMmedium containing no aromatic amino acid, 2% glucose and 1.0 mMtyrosine. These cultures were returned to the shaker (250 rpm, 30° C.)and samples (1.0 mL) were taken for HPLC analysis after 2.0, 4.0, 6.0and 16 h. Results are shown in Table 14.

TABLE 14 Effect of Aromatic Amino Acids on PHCA Production of AR040SWYeast Strain PHCA production (μM) 2.0 h 4.0 h 6.0h o/n no aromatic aa 00.609 0.806 1.327 Phe 0 0.872 1.147 1.735 Tyr 1.141 1.563 2.433 4.272

As is seen by the data in Table 14, when cells were starved for aromaticamino acids, no significant PHCA was produced. Addition of phenylalaninedid not have any significant effect on the level of PHCA produced.However, addition of tyrosine, resulted in significant increase in thelevel of PHCA. The results therefore confirm that the novel recombinantstrain containing the modified PAL gene developed in this inventionpreferred tyrosine as the substrate for PHCA production.

Example 15

Transformation and Expression of Mutant PAL/TAL in Maize and productionof PHCA

A chimeric gene comprising the mutant PAL/TAL gene (SEQ ID NO:8) insense orientation can be constructed by polymerase chain reaction (PCR)of the gene using appropriate oligonucleotide primers. Cloning sites(NcoI or SmaI) can be incorporated into the oligonucleotides to provideproper orientation of the DNA fragment when inserted into the digestedvector pML 103 as described below. Amplification is then performed in a100 μL volume in a standard PCR mix consisting of 0.4 mM of eacholigonucleotide and 0.3 pM of target DNA in 10 mM Tris-HCl, pH 8.3, 50mM KCl, 1.5 mM MgCl₂, 200 mM dGTP, 200 mM dATP, 200 mM dTTP, 200 mM dCTPand 0.025 unit DNA polymerase. Reactions are carried out in aPerkin-Elmer Cetus Thermocycler™ for 30 cycles comprising 1 min at 95°C., 2 min at 55° C. and 3 min at 72° C., with a final 7 min extension at72° C. after the last cycle. The amplified DNA is then digested withrestriction enzymes NcoI and Smal and fractionated on a 0.7% low meltingpoint agarose gel in 40 mM Tris-acetate, pH 8.5, 1 mM EDTA. Theappropriate band can be excised from the gel, melted at 68° C. andcombined with a 4.9 kb NcoI-Smal fragment of the plasmid pML103. PlasmidpML103 has been deposited under the terms of the Budapest Treaty withthe ATCC and bears accession number ATCC 97366. The DNA segment frompML103 contains a 1.05 kb SalI-NcoI promoter fragment of the maize 27 kDzein gene and a 0.96 kb Smal-SalI fragment from the 3′ end of the maize10 kD zein gene in the vector pGem9Zf(+) (Promega Corp., 7113 BenhartDr., Raleigh, N.C.). Vector and insert DNA can be ligated at 15° C.overnight, essentially as described (Maniatis). The ligated DNA may thenbe used to transform E. coli XL1-Blue (Epicurian Coli XL-1; Stratagene).Bacterial transformants can be screened by restriction enzyme digestionof plasmid DNA and limited nucleotide sequence analysis using thedideoxy chain termination method (DNA Sequencing Kit, U.S. Biochemical).The resulting plasmid construct would comprise a chimeric gene encoding,in the 5′ to 3′ direction, the maize 27 kD zein promoter, a DNA fragmentencoding the mutant PAL/TAL enzyme, and the 10 kD zein 3′ region.

The chimeric gene so constructed can then be introduced into corn cellsby the following procedure. Immature corn embryos can be dissected fromdeveloping caryopses derived from crosses of the inbred corn lines H99and LH132 (Indiana Agric. Exp. Station, IN, USA). The embryos areisolated 10 to 11 days after pollination when they are 1.0 to 1.5 mmlong. The embryos are then placed with the axis-side facing down and incontact with agarose-solidified N6 medium (Chu et al., Sci. Sin. Peking18:659-668 (1975)). The embryos are kept in the dark at 27° C. Friableembryogenic callus consisting of undifferentiated masses of cells withsomatic proembryoids and embryoids borne on suspensor structuresproliferates from the scutellum of these immature embryos. Theembryogenic callus isolated from the primary explant can be cultured onN6 medium and sub-cultured on this medium every 2 to 3 weeks. Theplasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag, vFrankfurt, Germany), may be used in transformation experiments in orderto provide for a selectable marker. This plasmid contains the Pat gene(see European Patent Publication 0 242 236) which encodesphosphinothricin acetyl transferase (PAT). The enzyme PAT confersresistance to herbicidal glutamine synthetase inhibitors such asphosphinothricin. The pat gene in p35S/Ac is under the control of the35S promoter from Cauliflower Mosaic Virus (Odell et al., Nature313:810-812 (1985)) and the 3M region of the nopaline synthase gene fromthe T-DNA of the Ti plasmid of Agrobacterium tumefaciens. The particlebombardment method (Klein et al., Nature 327:70-73 (1987)) may be usedto transfer genes to the callus culture cells. According to this method,gold particles (1 μm in diameter) are coated with DNA using thefollowing technique. Ten μg of plasmid DNAs are added to 50 μL of asuspension of gold particles (60 mg per mL). Calcium chloride (50 μL ofa 2.5 M solution) and spermidine free base (20 μL of a 1.0 M solution)are added to the particles. The suspension is vortexed during theaddition of these solutions. After 10 min, the tubes are brieflycentrifuged (5 sec at 15,000 rpm) and the supernatant removed. Theparticles are resuspended in 200 μL of absolute ethanol, centrifugedagain and the supernatant removed. The ethanol rinse is performed againand the particles resuspended in a final volume of 30 μL of ethanol. Analiquot (5 μL) of the DNA-coated gold particles can be placed in thecenter of a flying disc (Bio-Rad Labs, 861 Ridgeview Dr, Medina, Ohio).The particles are then accelerated into the corn tissue with a PDS-1000/He (Bio-Rad Labs, 861 Ridgeview Dr., Medina, Ohio), using a heliumpressure of 1000 psi, a gap distance of 0.5 cm and a flying distance of1.0 cm.

For bombardment, the embryogenic tissue is placed on filter paper overagarose-solidified N6 medium. The tissue is arranged as a thin lawn andcovers a circular area of about 5 cm in diameter. The petri dishcontaining the tissue can be placed in the chamber of the PDS-1000/Heapproximately 8 cm from the stopping screen. The air in the chamber isthen evacuated to a vacuum of 28 inches of Hg. The macrocarrier isaccelerated with a helium shock wave using a rupture membrane thatbursts when the He pressure in the shock tube reaches 1000 psi.

Seven days after bombardment the tissue can be transferred to N6 mediumthat contains gluphosinate (2 mg per liter) and lacks casein or proline.The tissue continues to grow slowly on this medium. After an additional2 weeks, the tissue can be transferred to fresh N6 medium containinggluphosinate. After 6 weeks, areas of about 1 cm in diameter of activelygrowing callus can be identified on some of the plates containing thegluphosinate-supplemented medium. These calli may continue to grow whensub-cultured on the selective medium. Plants can be regenerated from thetransgenic callus by first transferring clusters of tissue to N6 mediumsupplemented with 0.2 mg per liter of 2,4-D. After two weeks, the tissuecan be transferred to regeneration medium (Fromm et al., Bio/Technology8:833-839 (1990)).

Levels of PHCA production is expected to range from about 0.1% to about10% dry weight of the plant tissue.

Example 16

Selection For An Improved TAL Enzyme Using L-Tyrosine As A Sole CarbonSource

The mutagenized TAL gene (SEQ ID NO:8) is introduced into anAcinetobacter chromosome by natural transformation, essentially asdescribed by Kok et al., (Appl. Environ. Microbiol. 65:1675-1680(1999)), incorporated herein by reference. The TAL gene is inserted inthe host in a vector under the control a constitutive promoter and inthe presence of an antibiotic resistance marker gene. Transformants arecultured on an M9 salt media (Example 4) containing 15 g agar, 6 gNa₂HPO₄, 3 g KH₂PO₄, 0.5 g NaCl, 1 g NH₄Cl, 0.5 g L-tyrosine, 2 ml 1 MMgSO₄, 0.1 ml 1 M CaCl₂ in 1 L distilled water (pH 7.4). Transformantsare isolated on the basis of antibiotic resistance. Transformantscontaining an evolved TAL gene that improves the conversion ofL-tyrosine to PHCA show better growth on minimal media containingL-tyrosine and form larger colonies. These larger colonies are recoveredfor additional rounds of evolution until the desired level of TALactivity is achieved.

14 1 34 DNA Artificial Sequence Description of Artificial Sequenceprimer 1 atagtagaat tcatggcacc ctcgctcgac tcga 34 2 29 DNA ArtificialSequence Description of Artificial Sequence primer 2 gagagactgcagagaggcag ccaagaacg 29 3 45 DNA Artificial Sequence Description ofArtificial Sequence primer 3 gagagactcg agcccgggag atctcagacc aagtttactcatata 45 4 45 DNA Artificial Sequence Description of Artificial Sequenceprimer 4 gagagactcg agctgcagtc tagaactctt ttttcaatat tattg 45 5 22 DNAArtificial Sequence Description of Artificial Sequence primer 5tagctctaga atggcaccct cg 22 6 21 DNA Artificial Sequence Description ofArtificial Sequence primer 6 aactgcagct aagcgagcat c 21 7 2151 DNARhodotorula glutinis 7 atggcaccct cgctcgactc gatctcgcac tcgttcgcaaacggcgtcgc atccgcaaag 60 caggctgtca atggcgcctc gaccaacctc gcagtcgcaggctcgcacct gcccacaacc 120 caggtcacgc aggtcgacat cgtcgagaag atgctcgccgcgccgaccga ctcgacgctc 180 gaactcgacg gctactcgct caacctcgga gacgtcgtctcggccgcgag gaagggcagg 240 cctgtccgcg tcaaggacag cgacgagatc cgctcaaagattgacaaatc ggtcgagttc 300 ttgcgctcgc aactctccat gagcgtctac ggcgtcacgactggatttgg cggatccgca 360 gacacccgca ccgaggacgc catctcgctc cagaaggctctcctcgagca ccagctctgc 420 ggtgttctcc cttcgtcgtt cgactcgttc cgcctcggccgcggtctcga gaactcgctt 480 cccctcgagg ttgttcgcgg cgccatgaca atccgcgtcaacagcttgac ccgcggccac 540 tcggctgtcc gcctcgtcgt cctcgaggcg ctcaccaacttcctcaacca cggcatcacc 600 cccatcgtcc ccctccgcgg caccatctct gcgtcgggcgacctgtctcc tctctcctac 660 attgcagcgg ccatcagcgg tcacccggac agcaaggtgcacgtcgtcca cgagggcaag 720 gagaagatcc tgtacgcccg cgaggcgatg gcgctcttcaacctcgagcc cgtcgtcctc 780 ggcccgaagg aaggtctcgg tctcgtcaac ggcaccgccgtctcagcatc gatggccacc 840 ctcgctctgc acgacgctca catgctctcg ctcctctcgcagtcgctcac ggccatgacg 900 gtcgaagcga tggtcggcca cgccggctcg ttccaccccttccttcacga cgtcacgcgc 960 cctcacccga cgcagatcga agtcgcggga aacatccgcaagctcctcga gggaagccgc 1020 tttgctgtcc accatgagga ggaggtcaag gtcaaggacgacgagggcat tctccgccag 1080 gaccgctacc ccttgcgcac gtctcctcag tggctcggcccgctcgtcag cgacctcatt 1140 cacgcccacg ccgtcctcac catcgaggcc ggccagtcgacgaccgacaa ccctctcatc 1200 gacgtcgaga acaagacttc gcaccacggc ggcaatttccaggctgccgc tgtggccaac 1260 accatggaga agactcgcct cgggctcgcc cagatcggcaagctcaactt cacgcagctc 1320 accgagatgc tcaacgccgg catgaaccgc ggcctcccctcctgcctcgc ggccgaagac 1380 ccctcgctct cctaccactg caagggcctc gacatcgccgctgcggcgta cacctcggag 1440 ttgggacacc tcgccaaccc tgtgacgacg catgtccagccggctgagat ggcgaaccag 1500 gcggtcaact cgcttgcgct catctcggct cgtcgcacgaccgagtccaa cgacgtcctt 1560 tctctcctcc tcgccaccca cctctactgc gttctccaagccatcgactt gcgcgcgatc 1620 gagttcgagt tcaagaagca gttcggccca gccatcgtctcgctcatcga ccagcacttt 1680 ggctccgcca tgaccggctc gaacctgcgc gacgagctcgtcgagaaggt gaacaagacg 1740 ctcgccaagc gcctcgagca gaccaactcg tacgacctcgtcccgcgctg gcacgacgcc 1800 ttctccttcg ccgccggcac cgtcgtcgag gtcctctcgtcgacgtcgct ctcgctcgcc 1860 gccgtcaacg cctggaaggt cgccgccgcc gagtcggccatctcgctcac ccgccaagtc 1920 cgcgagacct tctggtccgc cgcgtcgacc tcgtcgcccgcgctctcgta cctctcgccg 1980 cgcactcaga tcctctacgc cttcgtccgc gaggagcttggcgtcaaggc ccgccgcgga 2040 gacgtcttcc tcggcaagca agaggtgacg atcggctcgaacgtctccaa gatctacgag 2100 gccatcaagt cgggcaggat caacaacgtc ctcctcaagatgctcgctta g 2151 8 716 PRT Rhodotorula glutinis 8 Met Ala Pro Ser LeuAsp Ser Ile Ser His Ser Phe Ala Asn Gly Val 1 5 10 15 Ala Ser Ala LysGln Ala Val Asn Gly Ala Ser Thr Asn Leu Ala Val 20 25 30 Ala Gly Ser HisLeu Pro Thr Thr Gln Val Thr Gln Val Asp Ile Val 35 40 45 Glu Lys Met LeuAla Ala Pro Thr Asp Ser Thr Leu Glu Leu Asp Gly 50 55 60 Tyr Ser Leu AsnLeu Gly Asp Val Val Ser Ala Ala Arg Lys Gly Arg 65 70 75 80 Pro Val ArgVal Lys Asp Ser Asp Glu Ile Arg Ser Lys Ile Asp Lys 85 90 95 Ser Val GluPhe Leu Arg Ser Gln Leu Ser Met Ser Val Tyr Gly Val 100 105 110 Thr ThrGly Phe Gly Gly Ser Ala Asp Thr Arg Thr Glu Asp Ala Ile 115 120 125 SerLeu Gln Lys Ala Leu Leu Glu His Gln Leu Cys Gly Val Leu Pro 130 135 140Ser Ser Phe Asp Ser Phe Arg Leu Gly Arg Gly Leu Glu Asn Ser Leu 145 150155 160 Pro Leu Glu Val Val Arg Gly Ala Met Thr Ile Arg Val Asn Ser Leu165 170 175 Thr Arg Gly His Ser Ala Val Arg Leu Val Val Leu Glu Ala LeuThr 180 185 190 Asn Phe Leu Asn His Gly Ile Thr Pro Ile Val Pro Leu ArgGly Thr 195 200 205 Ile Ser Ala Ser Gly Asp Leu Ser Pro Leu Ser Tyr IleAla Ala Ala 210 215 220 Ile Ser Gly His Pro Asp Ser Lys Val His Val ValHis Glu Gly Lys 225 230 235 240 Glu Lys Ile Leu Tyr Ala Arg Glu Ala MetAla Leu Phe Asn Leu Glu 245 250 255 Pro Val Val Leu Gly Pro Lys Glu GlyLeu Gly Leu Val Asn Gly Thr 260 265 270 Ala Val Ser Ala Ser Met Ala ThrLeu Ala Leu His Asp Ala His Met 275 280 285 Leu Ser Leu Leu Ser Gln SerLeu Thr Ala Met Thr Val Glu Ala Met 290 295 300 Val Gly His Ala Gly SerPhe His Pro Phe Leu His Asp Val Thr Arg 305 310 315 320 Pro His Pro ThrGln Ile Glu Val Ala Gly Asn Ile Arg Lys Leu Leu 325 330 335 Glu Gly SerArg Phe Ala Val His His Glu Glu Glu Val Lys Val Lys 340 345 350 Asp AspGlu Gly Ile Leu Arg Gln Asp Arg Tyr Pro Leu Arg Thr Ser 355 360 365 ProGln Trp Leu Gly Pro Leu Val Ser Asp Leu Ile His Ala His Ala 370 375 380Val Leu Thr Ile Glu Ala Gly Gln Ser Thr Thr Asp Asn Pro Leu Ile 385 390395 400 Asp Val Glu Asn Lys Thr Ser His His Gly Gly Asn Phe Gln Ala Ala405 410 415 Ala Val Ala Asn Thr Met Glu Lys Thr Arg Leu Gly Leu Ala GlnIle 420 425 430 Gly Lys Leu Asn Phe Thr Gln Leu Thr Glu Met Leu Asn AlaGly Met 435 440 445 Asn Arg Gly Leu Pro Ser Cys Leu Ala Ala Glu Asp ProSer Leu Ser 450 455 460 Tyr His Cys Lys Gly Leu Asp Ile Ala Ala Ala AlaTyr Thr Ser Glu 465 470 475 480 Leu Gly His Leu Ala Asn Pro Val Thr ThrHis Val Gln Pro Ala Glu 485 490 495 Met Ala Asn Gln Ala Val Asn Ser LeuAla Leu Ile Ser Ala Arg Arg 500 505 510 Thr Thr Glu Ser Asn Asp Val LeuSer Leu Leu Leu Ala Thr His Leu 515 520 525 Tyr Cys Val Leu Gln Ala IleAsp Leu Arg Ala Ile Glu Phe Glu Phe 530 535 540 Lys Lys Gln Phe Gly ProAla Ile Val Ser Leu Ile Asp Gln His Phe 545 550 555 560 Gly Ser Ala MetThr Gly Ser Asn Leu Arg Asp Glu Leu Val Glu Lys 565 570 575 Val Asn LysThr Leu Ala Lys Arg Leu Glu Gln Thr Asn Ser Tyr Asp 580 585 590 Leu ValPro Arg Trp His Asp Ala Phe Ser Phe Ala Ala Gly Thr Val 595 600 605 ValGlu Val Leu Ser Ser Thr Ser Leu Ser Leu Ala Ala Val Asn Ala 610 615 620Trp Lys Val Ala Ala Ala Glu Ser Ala Ile Ser Leu Thr Arg Gln Val 625 630635 640 Arg Glu Thr Phe Trp Ser Ala Ala Ser Thr Ser Ser Pro Ala Leu Ser645 650 655 Tyr Leu Ser Pro Arg Thr Gln Ile Leu Tyr Ala Phe Val Arg GluGlu 660 665 670 Leu Gly Val Lys Ala Arg Arg Gly Asp Val Phe Leu Gly LysGln Glu 675 680 685 Val Thr Ile Gly Ser Asn Val Ser Lys Ile Tyr Glu AlaIle Lys Ser 690 695 700 Gly Arg Ile Asn Asn Val Leu Leu Lys Met Leu Ala705 710 715 9 2151 DNA Artificial Sequence Description of ArtificialSequence mutant from Rhodotorula glutinis 9 atggcaccct cgctcgactcgatctcgcac tcgttcgcaa acggcgtcgc atccgcaaag 60 caggctgtca atggcgcctcgaccaacctc gcagtcgcag gctcgcacct gcccacaacc 120 caggtcacgc aggtcgacatcgtcgagaag atgctcgccg cgccgaccga ctcgacgctc 180 gaactcgacg gctactcgctcaacctcgga gacgtcgtct cggccgcgag gaagggcagg 240 cctgtccgcg tcaaggacagcgacgagatc cgctcaaaga ttgacaaatc ggtcgagttc 300 ttgcgctcgc aactctccatgagcgtctac ggcgtcacga ctggatttgg cggatccgca 360 gacacccgca ccgaggacgccatctcgctc cagaaggctc tcctcgagca ccagctctgc 420 ggtgttctcc cttcgtcgttcgactcgttc cgcctcggcc gcggtctcga gaactcgctt 480 cccctcgagg ttgttcgcggcgccatgaca atccgcgtca acagcttgac ccgcggccac 540 tcggctgtcc gcctcgtcgtcctcgaggcg ctcaccaact tcctcaacca cggcatcacc 600 cccatcgtcc ccctccgcggcaccatctct gcgtcgggcg acctctctcc tctctcctac 660 attgcagcgg ccatcagcggtcacccggac agcaaggtgc acgtcgtcca cgagggcaag 720 gagaagatcc tgtacgcccgcgaggcgatg gcgctcttca acctcgagcc cgtcgtcctc 780 ggcccgaagg agggtctcggtctcgtcaac ggcaccgccg tctcagcatc gatggccacc 840 ctcgctctgc acgacgcacacatgctctcg ctcctctcgc agtcgctcac ggccatgacg 900 gtcgaagcga tggtcggccacgccggctcg ttccacccct tccttcacga cgtcacgcgc 960 cctcacccga cgcagatcgaagtcgcggga aacatccgca agctcctcga gggaagccgc 1020 tttgctgtcc accatgaggaggaggtcaag gtcaaggacg acgagggcat tctccgccag 1080 gaccgctacc ccttgcgcacgtctcctcag tggctcggcc cgctcgtcag cgacctcatt 1140 cacgcccacg ccgtcctcaccatcgaggcc ggccagtcga cgaccgacaa ccctctcatc 1200 gacgtcgaga acaagacttcgcaccacggc ggcaatttcc aggctgccgc tgtggccaac 1260 accatggaga agactcgcctcgggctcgcc cagatcggca agctcaactt cacgcagctc 1320 accgagatgc tcaacgccggcatgaaccgc ggcctcccct cctgcctcgc ggccgaagac 1380 ccctcgctct cctaccactgcaagggcctc gacatcgccg ctgcggcgta cacctcggag 1440 ttgggacacc tcgccaaccctgtgacgacg catgtccagc cggctgagat ggcgaaccag 1500 gcggtcaact cgcttgcgctcatctcggct cgtcgcacga ccgagtccaa cgacgtcctt 1560 tctctcctcc tcgccacccacctctactgc gttctccaag ccatcgactt gcgcgcgacc 1620 gagttcgagt tcaagaagcagttcggccca gccatcgtct cgctcatcga ccagcacttt 1680 ggctccgcca tgaccggctcgaacctgcgc gacgagctcg tcgagaaggt gaacaagacg 1740 ctcgccaagc gcctcgagcagaccaactcg tacgacctcg tcccgcgctg gcacgacgcc 1800 ttctccttcg ccgccggcaccgtcgtcgag gtcctctcgt cgacgtcgct ctcgctcgcc 1860 gccgtcaacg cctggaaggtcgccgccgcc gagtcggcca tctcgctcac ccgccaagtc 1920 cgcgagacct tctggtccgccgcgtcgacc tcgtcgcccg cgctctcgta cctctcgccg 1980 cgcactcaga tcctctacgccttcgtccgc gaggagcttg gcgtcaaggc ccgccgcgga 2040 gacgtcttcc tcggcaagcaagaggtgacg atcggctcga acgtctccaa gatctacgag 2100 gccatcaagt cgggcaggatcaacaacgtc ctcctcaaga tgctcgctta g 2151 10 716 PRT Artificial SequenceDescription of Artificial Sequence mutant from Rhodtorula glutinis 10Met Ala Pro Ser Leu Asp Ser Ile Ser His Ser Phe Ala Asn Gly Val 1 5 1015 Ala Ser Ala Lys Gln Ala Val Asn Gly Ala Ser Thr Asn Leu Ala Val 20 2530 Ala Gly Ser His Leu Pro Thr Thr Gln Val Thr Gln Val Asp Ile Val 35 4045 Glu Lys Met Leu Ala Ala Pro Thr Asp Ser Thr Leu Glu Leu Asp Gly 50 5560 Tyr Ser Leu Asn Leu Gly Asp Val Val Ser Ala Ala Arg Lys Gly Arg 65 7075 80 Pro Val Arg Val Lys Asp Ser Asp Glu Ile Arg Ser Lys Ile Asp Lys 8590 95 Ser Val Glu Phe Leu Arg Ser Gln Leu Ser Met Ser Val Tyr Gly Val100 105 110 Thr Thr Gly Phe Gly Gly Ser Ala Asp Thr Arg Thr Glu Asp AlaIle 115 120 125 Ser Leu Gln Lys Ala Leu Leu Glu His Gln Leu Cys Gly ValLeu Pro 130 135 140 Ser Ser Phe Asp Ser Phe Arg Leu Gly Arg Gly Leu GluAsn Ser Leu 145 150 155 160 Pro Leu Glu Val Val Arg Gly Ala Met Thr IleArg Val Asn Ser Leu 165 170 175 Thr Arg Gly His Ser Ala Val Arg Leu ValVal Leu Glu Ala Leu Thr 180 185 190 Asn Phe Leu Asn His Gly Ile Thr ProIle Val Pro Leu Arg Gly Thr 195 200 205 Ile Ser Ala Ser Gly Asp Leu SerPro Leu Ser Tyr Ile Ala Ala Ala 210 215 220 Ile Ser Gly His Pro Asp SerLys Val His Val Val His Glu Gly Lys 225 230 235 240 Glu Lys Ile Leu TyrAla Arg Glu Ala Met Ala Leu Phe Asn Leu Glu 245 250 255 Pro Val Val LeuGly Pro Lys Glu Gly Leu Gly Leu Val Asn Gly Thr 260 265 270 Ala Val SerAla Ser Met Ala Thr Leu Ala Leu His Asp Ala His Met 275 280 285 Leu SerLeu Leu Ser Gln Ser Leu Thr Ala Met Thr Val Glu Ala Met 290 295 300 ValGly His Ala Gly Ser Phe His Pro Phe Leu His Asp Val Thr Arg 305 310 315320 Pro His Pro Thr Gln Ile Glu Val Ala Gly Asn Ile Arg Lys Leu Leu 325330 335 Glu Gly Ser Arg Phe Ala Val His His Glu Glu Glu Val Lys Val Lys340 345 350 Asp Asp Glu Gly Ile Leu Arg Gln Asp Arg Tyr Pro Leu Arg ThrSer 355 360 365 Pro Gln Trp Leu Gly Pro Leu Val Ser Asp Leu Ile His AlaHis Ala 370 375 380 Val Leu Thr Ile Glu Ala Gly Gln Ser Thr Thr Asp AsnPro Leu Ile 385 390 395 400 Asp Val Glu Asn Lys Thr Ser His His Gly GlyAsn Phe Gln Ala Ala 405 410 415 Ala Val Ala Asn Thr Met Glu Lys Thr ArgLeu Gly Leu Ala Gln Ile 420 425 430 Gly Lys Leu Asn Phe Thr Gln Leu ThrGlu Met Leu Asn Ala Gly Met 435 440 445 Asn Arg Gly Leu Pro Ser Cys LeuAla Ala Glu Asp Pro Ser Leu Ser 450 455 460 Tyr His Cys Lys Gly Leu AspIle Ala Ala Ala Ala Tyr Thr Ser Glu 465 470 475 480 Leu Gly His Leu AlaAsn Pro Val Thr Thr His Val Gln Pro Ala Glu 485 490 495 Met Ala Asn GlnAla Val Asn Ser Leu Ala Leu Ile Ser Ala Arg Arg 500 505 510 Thr Thr GluSer Asn Asp Val Leu Ser Leu Leu Leu Ala Thr His Leu 515 520 525 Tyr CysVal Leu Gln Ala Ile Asp Leu Arg Ala Thr Glu Phe Glu Phe 530 535 540 LysLys Gln Phe Gly Pro Ala Ile Val Ser Leu Ile Asp Gln His Phe 545 550 555560 Gly Ser Ala Met Thr Gly Ser Asn Leu Arg Asp Glu Leu Val Glu Lys 565570 575 Val Asn Lys Thr Leu Ala Lys Arg Leu Glu Gln Thr Asn Ser Tyr Asp580 585 590 Leu Val Pro Arg Trp His Asp Ala Phe Ser Phe Ala Ala Gly ThrVal 595 600 605 Val Glu Val Leu Ser Ser Thr Ser Leu Ser Leu Ala Ala ValAsn Ala 610 615 620 Trp Lys Val Ala Ala Ala Glu Ser Ala Ile Ser Leu ThrArg Gln Val 625 630 635 640 Arg Glu Thr Phe Trp Ser Ala Ala Ser Thr SerSer Pro Ala Leu Ser 645 650 655 Tyr Leu Ser Pro Arg Thr Gln Ile Leu TyrAla Phe Val Arg Glu Glu 660 665 670 Leu Gly Val Lys Ala Arg Arg Gly AspVal Phe Leu Gly Lys Gln Glu 675 680 685 Val Thr Ile Gly Ser Asn Val SerLys Ile Tyr Glu Ala Ile Lys Ser 690 695 700 Gly Arg Ile Asn Asn Val LeuLeu Lys Met Leu Ala 705 710 715 11 1620 DNA Helianthus tuberosus unsure(1588) N = A,T,G,C 11 aaatcacaca acaccaccac caccgtaacc atggacctcctcctcataga aaaaaccctc 60 gtcgccttat tcgccgccat tatcggcgca atactaatctccaaactccg cggtaaaaaa 120 ttcaagctcc cacctggccc aatcccggtt ccaattttcggcaactggct acaagttggc 180 gatgatttga accaccggaa cttaaccgat ctggctaagaggtttggtga gatcttgctg 240 ctacgcatgg ggcagaggaa tctggtagtt gtgtcttcgcctgagcttgc taaagaggtg 300 ttgcatacac aaggagtgga gtttggttcg agaacaaggaatgttgtgtt cgatattttt 360 actgggaagg gtcaggatat ggtgtttacg gtttatggtgagcattggag gaagatgagg 420 aggatcatga ccgtaccctt tttcaccaac aaagttgttcagcaatacag gtatgggtgg 480 gaggctgagg ccgcggcggt tgtggacgat gtgaagaagaatccggctgc agcaactgaa 540 ggaatcgtga tccgaagacg gttacaactc atgatgtataacaacatgtt cagaatcatg 600 ttcgacagac gattcgaaag tgaagatgat cccttgtttttgaaactcaa ggcgttgaac 660 ggtgagagga gtcgattggc gcagagcttt gagtacaactatggcgattt catccctatt 720 ttgcggccgt ttttgagaaa ttatttgaag ttgtgcaaggaagttaaaga taaaaggatt 780 cagctcttca aggattactt cgttgacgaa aggaagaagattggaagcac taagaaaatg 840 gacaacaatc agttgaaatg tgccattgat cacattcttgaagctaaaga gaagggtgag 900 atcaatgaag acaatgttct ttacattgtt gaaaacatcaatgttgcagc aatcgagaca 960 actctatggt cgatcgaatg gggaattgcg gagctagttaaccatcccga gatccaagcc 1020 aaactcaggc acgagctcga caccaagctc gggcccggtgtccagatcac cgagcccgac 1080 gtccaaaacc tcccttacct ccaagccgtg gtcaaggaaaccctccgtct ccgtatggcg 1140 atcccgcttc tagtcccaca catgaacctc catgacgctaagctcggcgg gtttgacatc 1200 ccggccgaaa gcaagatctt ggtcaacgcg tggtggttagcaaacaaccc cgaccaatgg 1260 aagaaacccg aggagtttag gccagagagg tttttggaagaggaagcgaa ggttgaggct 1320 aacgggaatg attttaggta cttgccgttt ggagtcgggagaaggagttg ccccgggatt 1380 attcttgcat tgccgatact tggtattaca atcgggcgtttggtgcagaa tttcgagctg 1440 ttgcctccac cgggacagtc taagatcgat accgatgagaagggtgggca gtttagtttg 1500 catatcttga agcactctac tatcgtagct aaacctaggtcattttaagg attcttgttt 1560 atgttcttta ttgtatgata aaccaagngg ngnnggngnngngngannaa aaaaaaaaaa 1620 12 505 PRT Helianthus tuberosus 12 Met AspLeu Leu Leu Ile Glu Lys Thr Leu Val Ala Leu Phe Ala Ala 1 5 10 15 IleIle Gly Ala Ile Leu Ile Ser Lys Leu Arg Gly Lys Lys Phe Lys 20 25 30 LeuPro Pro Gly Pro Ile Pro Val Pro Ile Phe Gly Asn Trp Leu Gln 35 40 45 ValGly Asp Asp Leu Asn His Arg Asn Leu Thr Asp Leu Ala Lys Arg 50 55 60 PheGly Glu Ile Leu Leu Leu Arg Met Gly Gln Arg Asn Leu Val Val 65 70 75 80Val Ser Ser Pro Glu Leu Ala Lys Glu Val Leu His Thr Gln Gly Val 85 90 95Glu Phe Gly Ser Arg Thr Arg Asn Val Val Phe Asp Ile Phe Thr Gly 100 105110 Lys Gly Gln Asp Met Val Phe Thr Val Tyr Gly Glu His Trp Arg Lys 115120 125 Met Arg Arg Ile Met Thr Val Pro Phe Phe Thr Asn Lys Val Val Gln130 135 140 Gln Tyr Arg Tyr Gly Trp Glu Ala Glu Ala Ala Ala Val Val AspAsp 145 150 155 160 Val Lys Lys Asn Pro Ala Ala Ala Thr Glu Gly Ile ValIle Arg Arg 165 170 175 Arg Leu Gln Leu Met Met Tyr Asn Asn Met Phe ArgIle Met Phe Asp 180 185 190 Arg Arg Phe Glu Ser Glu Asp Asp Pro Leu PheLeu Lys Leu Lys Ala 195 200 205 Leu Asn Gly Glu Arg Ser Arg Leu Ala GlnSer Phe Glu Tyr Asn Tyr 210 215 220 Gly Asp Phe Ile Pro Ile Leu Arg ProPhe Leu Arg Asn Tyr Leu Lys 225 230 235 240 Leu Cys Lys Glu Val Lys AspLys Arg Ile Gln Leu Phe Lys Asp Tyr 245 250 255 Phe Val Asp Glu Arg LysLys Ile Gly Ser Thr Lys Lys Met Asp Asn 260 265 270 Asn Gln Leu Lys CysAla Ile Asp His Ile Leu Glu Ala Lys Glu Lys 275 280 285 Gly Glu Ile AsnGlu Asp Asn Val Leu Tyr Ile Val Glu Asn Ile Asn 290 295 300 Val Ala AlaIle Glu Thr Thr Leu Trp Ser Ile Glu Trp Gly Ile Ala 305 310 315 320 GluLeu Val Asn His Pro Glu Ile Gln Ala Lys Leu Arg His Glu Leu 325 330 335Asp Thr Lys Leu Gly Pro Gly Val Gln Ile Thr Glu Pro Asp Val Gln 340 345350 Asn Leu Pro Tyr Leu Gln Ala Val Val Lys Glu Thr Leu Arg Leu Arg 355360 365 Met Ala Ile Pro Leu Leu Val Pro His Met Asn Leu His Asp Ala Lys370 375 380 Leu Gly Gly Phe Asp Ile Pro Ala Glu Ser Lys Ile Leu Val AsnAla 385 390 395 400 Trp Trp Leu Ala Asn Asn Pro Asp Gln Trp Lys Lys ProGlu Glu Phe 405 410 415 Arg Pro Glu Arg Phe Leu Glu Glu Glu Ala Lys ValGlu Ala Asn Gly 420 425 430 Asn Asp Phe Arg Tyr Leu Pro Phe Gly Val GlyArg Arg Ser Cys Pro 435 440 445 Gly Ile Ile Leu Ala Leu Pro Ile Leu GlyIle Thr Ile Gly Arg Leu 450 455 460 Val Gln Asn Phe Glu Leu Leu Pro ProPro Gly Gln Ser Lys Ile Asp 465 470 475 480 Thr Asp Glu Lys Gly Gly GlnPhe Ser Leu His Ile Leu Lys His Ser 485 490 495 Thr Ile Val Ala Lys ProArg Ser Phe 500 505 13 1863 DNA Helianthus tuberosus 13 ttgtttgaagaagcgaaagc gcgatatgaa aaagctgtgt ttaaagtggt tgatttggat 60 gattatgctgctgatgatga ggagtatgca gagaaattca agaaggagac atttgctttc 120 ttcttcttggctacatatgg agatggtgag ccaactgata atgctgcaag attttataaa 180 tggttcaccgagggagatga taaaggagtt tggcttgaaa aacttcacta tggtgtgttt 240 ggtcttggcaacaaacagta tgagcatttc aacaagattg cattagtggt tgatgagggt 300 ctcacagagcagggtgcaaa gcgctttgtt ccagttggcc ttggagatga cgatcaatca 360 attgaagatgatttttctgc atggaaagaa ttagtgtggc ctgaattgga tcaattgctt 420 cttgatgaagacgacaagac tgctgccact ccttacacag ctgccattcc cgaataccga 480 gtcgtgtttcatgacaaacc tgatacgttt tccgagaatc atagtcaaac taatggtcat 540 actgttcacgatgctcaaca tccatgcaga tccaacgtgg ctgttaaaaa agagctccat 600 acccctgaatccgatcgctc ctgcactcat cttgaatttg acatctctca cactggacta 660 tcatacgaaactggggatca cgtcggtgtc tactgtgaaa acctaattga agtagtggag 720 gaagctgagaaactgatagg attaccagca gatacttatt tctcattaca cattgataac 780 gaagatggaacaccactcgg tggacctaca ttgcagcctc ctttccctcc ctgcacttta 840 agaaaagcattgaccaatta cgcagatctg ttgagttctc ccaaaaagtc aaccttgctt 900 gctctagctgcgcatgcttc tgatgccact gaagctgatc gactacaatt tcttgcatct 960 cgtgagggcaaggatgaata tgctgaatgg attgttgcaa accaaagaag ccttcttgag 1020 gtcatggaagcttttccgtc agctaaacct ccgctcgggg ttttctttgc agctattgcc 1080 ccgcgtttgcagcctcgata ctactctatt tcttcctccc caaagatggt acccaacagg 1140 attcatgttacgtgtgcatt agtttatgag aagactcctg gaggtcgtat ccacaaagga 1200 atatgctcaacctggatgaa gaatgctgtg cctttgaccg aaaatcaaga ttgcagctcg 1260 gcacccatttttgttagaac atcgaacttc agacttccag ctgaccctaa agtcccggtt 1320 atcatgattggccctggaac cgggttggct ccgtttagag gttttcttca agaaagatta 1380 gctctcaaggaatctggaac cgaactcggt caatccattt tgttcttcgg ttgcagaaac 1440 cgtaaagtggatttcatata tgagaatgaa ctgaacaact ttgttgaaaa tggcgcgctt 1500 tccgagcttgacatggcttt ctctcgcgaa ggcgcatcta aagaatacgt gcaacataaa 1560 atgagccaaaaggcttcgga tatatggaac atgctttctg agggagcata cttatacgtg 1620 tgtggtgatgccaaaggcat ggctaaagat gtacaccgaa cccttcacac cattgtgcaa 1680 gaacagggaaatttggattc ctctaaagca gagctgtatg tgaagaatct acaaatgtcg 1740 ggaagatacctccgtgatgt ttggtgatct atcgagtaaa acggaaataa atgtgagggg 1800 aatttataaacactagttta tgacagtata attttgatct tttacagtca gtaattcgaa 1860 ttt 1863 14588 PRT Helianthus tuberosus 14 Leu Phe Glu Glu Ala Lys Ala Arg Tyr GluLys Ala Val Phe Lys Val 1 5 10 15 Val Asp Leu Asp Asp Tyr Ala Ala AspAsp Glu Glu Tyr Ala Glu Lys 20 25 30 Phe Lys Lys Glu Thr Phe Ala Phe PhePhe Leu Ala Thr Tyr Gly Asp 35 40 45 Gly Glu Pro Thr Asp Asn Ala Ala ArgPhe Tyr Lys Trp Phe Thr Glu 50 55 60 Gly Asp Asp Lys Gly Val Trp Leu GluLys Leu His Tyr Gly Val Phe 65 70 75 80 Gly Leu Gly Asn Lys Gln Tyr GluHis Phe Asn Lys Ile Ala Leu Val 85 90 95 Val Asp Glu Gly Leu Thr Glu GlnGly Ala Lys Arg Phe Val Pro Val 100 105 110 Gly Leu Gly Asp Asp Asp GlnSer Ile Glu Asp Asp Phe Ser Ala Trp 115 120 125 Lys Glu Leu Val Trp ProGlu Leu Asp Gln Leu Leu Leu Asp Glu Asp 130 135 140 Asp Lys Thr Ala AlaThr Pro Tyr Thr Ala Ala Ile Pro Glu Tyr Arg 145 150 155 160 Val Val PheHis Asp Lys Pro Asp Thr Phe Ser Glu Asn His Ser Gln 165 170 175 Thr AsnGly His Thr Val His Asp Ala Gln His Pro Cys Arg Ser Asn 180 185 190 ValAla Val Lys Lys Glu Leu His Thr Pro Glu Ser Asp Arg Ser Cys 195 200 205Thr His Leu Glu Phe Asp Ile Ser His Thr Gly Leu Ser Tyr Glu Thr 210 215220 Gly Asp His Val Gly Val Tyr Cys Glu Asn Leu Ile Glu Val Val Glu 225230 235 240 Glu Ala Glu Lys Leu Ile Gly Leu Pro Ala Asp Thr Tyr Phe SerLeu 245 250 255 His Ile Asp Asn Glu Asp Gly Thr Pro Leu Gly Gly Pro ThrLeu Gln 260 265 270 Pro Pro Phe Pro Pro Cys Thr Leu Arg Lys Ala Leu ThrAsn Tyr Ala 275 280 285 Asp Leu Leu Ser Ser Pro Lys Lys Ser Thr Leu LeuAla Leu Ala Ala 290 295 300 His Ala Ser Asp Ala Thr Glu Ala Asp Arg LeuGln Phe Leu Ala Ser 305 310 315 320 Arg Glu Gly Lys Asp Glu Tyr Ala GluTrp Ile Val Ala Asn Gln Arg 325 330 335 Ser Leu Leu Glu Val Met Glu AlaPhe Pro Ser Ala Lys Pro Pro Leu 340 345 350 Gly Val Phe Phe Ala Ala IleAla Pro Arg Leu Gln Pro Arg Tyr Tyr 355 360 365 Ser Ile Ser Ser Ser ProLys Met Val Pro Asn Arg Ile His Val Thr 370 375 380 Cys Ala Leu Val TyrGlu Lys Thr Pro Gly Gly Arg Ile His Lys Gly 385 390 395 400 Ile Cys SerThr Trp Met Lys Asn Ala Val Pro Leu Thr Glu Asn Gln 405 410 415 Asp CysSer Ser Ala Pro Ile Phe Val Arg Thr Ser Asn Phe Arg Leu 420 425 430 ProAla Asp Pro Lys Val Pro Val Ile Met Ile Gly Pro Gly Thr Gly 435 440 445Leu Ala Pro Phe Arg Gly Phe Leu Gln Glu Arg Leu Ala Leu Lys Glu 450 455460 Ser Gly Thr Glu Leu Gly Gln Ser Ile Leu Phe Phe Gly Cys Arg Asn 465470 475 480 Arg Lys Val Asp Phe Ile Tyr Glu Asn Glu Leu Asn Asn Phe ValGlu 485 490 495 Asn Gly Ala Leu Ser Glu Leu Asp Met Ala Phe Ser Arg GluGly Ala 500 505 510 Ser Lys Glu Tyr Val Gln His Lys Met Ser Gln Lys AlaSer Asp Ile 515 520 525 Trp Asn Met Leu Ser Glu Gly Ala Tyr Leu Tyr ValCys Gly Asp Ala 530 535 540 Lys Gly Met Ala Lys Asp Val His Arg Thr LeuHis Thr Ile Val Gln 545 550 555 560 Glu Gln Gly Asn Leu Asp Ser Ser LysAla Glu Leu Tyr Val Lys Asn 565 570 575 Leu Gln Met Ser Gly Arg Tyr LeuArg Asp Val Trp 580 585

What is claimed is:
 1. A method for the production ofPara-hydroxycinnamic acid comprising: (i) contacting a recombinant hostcell with a fermentable carbon substrate, said recombinant cell lackinga cinnamate hydroxylase activity and comprising a gene encoding apolypeptide having tyrosine ammonia lyase activity operably linked tosuitable regulatory sequences; (ii) growing said recombinant cell for atime sufficient to produce Para-hydroxycinnamic acid; and (iii)optionally recovering said Para-hydroxycinnamic acid.
 2. A methodaccording to claim 1 wherein said fermentable carbon substrate isselected from the group consisting of monosaccharides, oligosaccharides,polysaccharides, carbon dioxide, methanol, formaldehyde, formate, andcarbon-containing amines.
 3. A method according to claim 2 wherein saidfermentable carbon substrate is glucose.
 4. A method according to claim1 wherein said recombinant host cell is selected from the groupconsisting of bacteria, yeasts, filamentous fungi, algae and plantcells.
 5. A method according to claim 4 wherein said recombinant hostcell is selected from the group consisting of Aspergillus, Arthrobotrys,Saccharomyces, Zygosaccharomyces, Pichia, Kluyveromyces, Candida,Hansenula, Debaryomyces, Mucor, Torulopsis, Methylobacter, Escherichia,Salmonella, Bacillus, Acinetobacter, Rhodococcus, Rhodobacter,Synechocystis, Streptomyces, and Pseudomonas.
 6. A method according toclaim 1 wherein said recombinant host cell is selected from the groupconsisting of soybean, rapeseed, sunflower, cotton, corn, tobacco,alfalfa, wheat, barley, oats, sorghum, rice, broccoli, cauliflower,cabbage, parsnips, melons, carrots, celery, parsley, tomatoes, potatoes,strawberries, peanuts, grapes, grass seed crops, sugar beets, sugarcane, beans, peas, rye, flax, hardwood trees, softwood trees, and foragegrasses.
 7. A method according to claim 1 wherein said tyrosine ammonialyase has a catalytic efficiency from about 4.14×10³ M⁻¹ sec⁻¹ to about1×10⁹ M⁻¹ sec⁻¹.
 8. A method according to claim 1 wherein said geneencoding a polypeptide having tyrosine ammonia lyase activity encodesthe polypeptide set forth in SEQ ID NO:8 or SEQ ID NO:10.
 9. A methodaccording to claim 1 wherein the gene encoding a polypeptide havingtyrosine ammonia lyase activity is derived from Rhodosporidium.
 10. Amethod according to claim 1 wherein said gene encoding a polypeptidehaving tyrosine ammonia lyase activity encodes the polypeptide set forthin SEQ ID NO:10.